AU743524B2

AU743524B2 - Chemiluminescent reactions using dihydroxyaromatic compounds and heterocyclic enol phosphates

Info

Publication number: AU743524B2
Application number: AU80576/98A
Authority: AU
Inventors: Hashem Akhavan-Tafti
Original assignee: Lumigen Inc
Current assignee: Lumigen Inc
Priority date: 1998-06-12
Filing date: 1998-06-12
Publication date: 2002-01-31
Anticipated expiration: 2018-06-12
Also published as: AU8057698A

Description

WO 99/40161 PCT/US98/11489 CHEMILUMINESCENT REACTIONS USING DIHYDROXYAROMATIC COMPOUNDS AND HETEROCYCLIC ENOL PHOSPHATES FIELD OF THE INVENTION The present invention relates to chemiluminescent methods and compositions for generating chemiluminescence.

In particular, the present invention relates to methods of generating chemiluminescence comprising reacting a dihydroxyaromatic compound and a heterocyclic enol phosphate compound in the presence of oxygen. The present invention also relates to chemiluminescent methods and compositions for generating chemiluminescence by the action of a hydrolytic enzyme. The methods for generating chemiluminescence comprise reacting a hydrolytic enzyme with a protected dihydroxy-aromatic compound in which one of the hydroxy groups is protected with an enzyme cleavable group to release the dihydroxyaromatic compound for reaction with a heterocyclic enol phosphate compound to produce chemiluminescence. The invention further relates to the use of the present chemiluminescent methods in assays for detecting analytes including hydrolytic enzymes and enzyme-labeled specific binding partners in specific binding pair assays including immunoassays, nucleic acid probe assays and the like.

BACKGROUND OF THE INVENTION 1. Chemiluminescent Detection of Hydrolytic Enzymes Hydrolytic enzymes such as alkaline phosphatase and 1- 1 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 galactosidase are frequently used as markers or labels in enzyme-linked assays for biological molecules and other analytes of interest such as drugs, hormones, steroids and cancer markers. In addition, phosphatase enzymes, e.g.

alkaline phosphatase (AP) and acid phosphatase (AcP), are clinically significant in their own right in human and veterinary diagnostics. Chemiluminescent detection of these enzymes offers a safe, convenient and sensitive means to provide a quantitative measure of the amount of enzyme in a sample or of the amount of an enzyme-labeled analyte or labeled specific binding partner for an analyte. Numerous chemiluminescent reaction schemes are known in the art for quantitating the level of particular hydrolytic enzymes.

Many of these schemes are complex and expensive, requiring multiple enzymes or several reagents. Commercial acceptance of such methods for large volume testing has been slow.

a. Chemiluminescent Reaction of Acridan ComDounds with Alkaline Phosphatase. PCT Application US97/00015, filed on January 15, 1997 describes a chemiluminescent reaction between a phosphatase enzyme and a compound comprising an heterocyclic ring group and an enol phosphate group. Also described is the improvement afforded by incorporation of a cationic aromatic compound in the chemiluminescent reaction between a phosphatase and the heterocyclic enol phosphate compound. In contrast to the present methods, the methods disclosed in these applications rely on the reaction of a phosphatase enzyme with this heterocyclic enol phosphate compound to cleave the phosphate group. In the present methods, no enzyme is used to cleave the phosphate group.

2 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 b. Reactions Involving the Generation of Reducing Agents. Chemiluminescent methods involving the generation of a reducing agent from a phosphate ester catalyzed by alkaline phosphatase have been reported. Maeda, A.

Tsuji, K.H. Yang, S. Kamada, Biolum. and Chemilum. Current Status, 119-22 (1991); M. Kitamura, M. Maeda, A. Tsuji, J.

Biolumin. Chemilumin., 10, 1-7 (1995); H. Sasamoto, M.

Maeda, A. Tsuji, Anal. Chim. Acta, 306, 161-6 (1995)). The reducing agent reacts with oxygen and lucigenin to produce light. Representative reducing agents include ascorbic acid, glycerol, NADH, dihydroxyacetone, cortisol and phenacyl alcohol. A PCT application, W096/04400, and a publication Arakawa, M. Maeda, A. Tsuji, Anal.

Biochem., 199, 238-242 (1991)) also describe chemiluminescent assays of alkaline phosphatase using indoxyl phosphate compounds and other related phosphate compounds which produce reducing species on dephosphorylation. Chemiluminescence is produced in the presence of lucigenin or other luminescence amplifying agents by virtue of their reaction with H 2 0 2 or superoxide ion.

U.S. Patent 5,589,328 to Mahant discloses a chemiluminescent reaction whereby indoxyl esters, thioindoxyl esters and benzofuran esters are hydrolyzed by an enzyme and thereby generate superoxide. Luminescence is amplified by adding a chemiluminescence generating reagent such as lucigenin. Lucigenin produces chemiluminescence by reaction with superoxide.

c. Enzvmaticallv Triqqerable Dioxetanes. Stable 1,2dioxetanes bearing a protected phenol triggering group 3 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 undergo a chemiluminescent decomposition upon removal of a protecting group P. Schaap, T. S. Chen, R. S. Handley, R. DeSilva, and B. P. Giri, Tetrahedron Lett., 1155 (1987); A. P. Schaap, R. S. Handley, and B. P. Giri, Tetrahedron Lett., 935 (1987); A. P. Schaap, M. D. Sandison, and R. S.

Handley, Tetrahedron Lett., 1159 (1987); and A. P. Schaap, Photochem. Photobiol., 47S, 50S (1988)). Such enzymatically triggerable dioxetanes are triggered by reaction with a hydrolytic enzyme to thereby accelerate the chemiluminescent decomposition rate of the dioxetane by orders of magnitude. Numerous examples of such triggerable dioxetanes are now known in the art. However, an inherent disadvantage of most triggerable dioxetanes is their tendency to generate background chemiluminescence in the absence of enzyme through slow thermal decomposition or non-enzymatic hydrolysis.

d. Luciferin Derivatives. Phosphate and galactoside derivatives of firefly luciferin are known Ugarova, Y.

Vosny, G. Kutuzova, I. Dementieva, Biolum. and Chemilum.

New Perspectives, P. Stanley and L.J. Kricka, eds., Wiley, Chichester, 511-4 (1981); W. Miska, R. Geiger, J. Biolumin.

Chemilumin., 4, 119-28 (1989)). Treatment of the firefly luciferin derivative with the appropriate enzyme liberates firefly luciferin which is reacted in a second step with luciferase and ATP to produce light.

e. Luminol Derivatives. A phosphate and a NAG derivative of luminol are known Sasamoto, Y. Ohkura, Chem. Pharm. Bull., 38, 1323-5 (1991); M. Nakazono, H.

Nohta, K. Sasamoto, Y. Ohkura, Anal. Sci., 8, 779-83 (1992)). Treatment of the luminol derivative with the 4 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 appropriate enzyme liberates luminol which is reacted in a subsequent step with ferricyanide to produce light.

f. Coupled Enzyme Methods. Applicants' PCT application WO 96/07911 discloses the enzymatic generation of a phenolic enhancer from a protected phenol compound using a hydrolytic enzyme. The phenol compound enhances the chemiluminescent oxidation of acridancarboxylic acid derivatives with a peroxidase enzyme. U.S. 5,306,621 discloses a similar coupled enzyme reaction where a dihydrophthalazinedione is used as the chemiluminescent peroxidase substrate. Protected dihydroxyaromatic compounds in which one of the hydroxy groups is protected with a group cleavable by a hydrolytic enzyme are not usable in these methods since dihydroxyaromatic compounds are not peroxidase enhancers. Numerous other chemiluminescent methods and assays for determining hydrolytic enzymes such as phosphatase enzymes through coupled enzyme reactions are known. A compilation of such methods is listed in A. Tsuji, M. Maeda, H. Arakawa, Anal. Sci., 5, 497-506 (1989). All of the coupled enzyme methods require two or more enzymes and utilize either luminol or a luminol analog, lucigenin or bacterial luciferin as the chemiluminescent species.

Many of the aforementioned methods suffer the drawback of requiring multiple reagents or enzymes in order to generate the luminescent signal. The added expense or operational complexity has hindered commercial acceptance of these methods in spite of their demonstrated exceptional detection sensitivity. Chemiluminescent methods for detecting and quantitating hydrolytic enzymes which achieve these levels of sensitivity but do not require additional SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 enzymes or auxiliary reagents in addition to the enzyme substrate would be advantageous. The present invention provides such methods and compounds.

2. Enzymatic Release of Electron-Transfer Agents (ETA) a.Colorimetric and Fluorimetric Detection U.S. Patent 4,952,495 discloses and claims methods for determination of hydrolytic enzymes by the enzymatic formation of a substance capable of mediating an electron-transfer reaction leading to the production of a detectable species.

The ETA can comprise in one embodiment a reducing substance such as a substituted hydroquinone compound. The detectable species form a colored or fluorescent product. It is further taught that the detectable species can be a shiftable detectable species comprising a quinone linked through a linking group to a chromogenic, fluorogenic or chemiluminogenic species. There is no teaching or suggestion of any chemiluminogenic detectable species other than such a shiftable compound. There is no suggestion or teaching of the presently described heterocyclic enol phosphate compounds.

b. Amerometric Detection of Alkaline Phosphatase (AP) A method is known for the amperometric detection of alkaline phosphatase comprising the enzymatic generation of hydroquinone from its monophosphate by AP, oxidation of the hydroquinone at the surface of an electrode to produce benzoquinone, regeneration of hydroquinone by the second enzyme glucose oxidase which transfers electrons from it substrate glucose. The quantity of AP is detected as an increase in electrical current.

6 SUBSTITUTE SHEET (RULE 26) P:\WPDOCS\Hjw\spec\7499280pgs.doc-09/I 1/01 SUMMARY OF THE INVENTION According to an aspect of the present invention there is provided a method of generating chemiluminescence by reacting a dihydroxyaromatic compound with a second compound containing a heterocyclic ring group and an enol phosphate group which undergoes a reaction in the presence of oxygen to produce a chemiluminescence.

According to another aspect of the present invention there is provided a method of generating chemiluminescence by the action of a hydrolytic enzyme comprising a) reacting the hydrolytic enzyme with a protected dihydroxyaromatic compound in which one of the hydroxy groups is protected with a group cleavable by the hydrolytic enzyme to produce a dihydroxyaromatic compound; and 15 b) reacting the dihydroxyaromatic compound with a heterocyclic enol phosphate compound to produce chemiluminescence.

According to a further aspect of the present invention there is provided a chemiluminescent composition comprising o 20 a dihydroxyaromatic compound and a heterocyclic enol phosphate compound. The present invention further provides :a composition which produces chemiluminescence upon reaction with a hydrolytic enzyme wherein the composition comprises a protected dihydroxyaromatic compound in which one of the hydroxy groups is protected with a group cleavable by the hydrolytic enzyme and a heterocyclic enol phosphate compound.

According to yet another aspect of the present invention there is provided methods for conducting an assay of an analyte comprising reacting a dihydroxyaromatic compound with a heterocyclic enol phosphate compound to produce chemiluminescence, measuring the chemiluminescence -7- P:\WPDOCSHjw\pcs\7499280pgs.doc-09/1 1/01 and relating the chemiluminescence to the amount of the analyte.

A further aspect involves a method of conducting an assay of an analyte comprising reacting a dihydroxyaromatic compound and a heterocyclic enol phosphate compound to produce chemiluminescence, measuring the chemiluminescence and relating the chemiluminescence to the amount to the analyte, wherein the heterocyclic enol phosphate compound is provided as a label on a specific binding pair member.

A further aspect involves a method of conducting an assay of an analyte comprising reacting a hydrolytic enzyme, a protected dihydroxyaromatic compound and a heterocyclic enol phosphate compound to produce chemiluminescence, measuring the chemiluminescence and relating the S 15 chemiluminescence to the amount of the analyte.

o In yet another aspect of the present invention there is provided a method of detecting a hydrolytic enzyme in a sample which comprises reacting the sample containing or suspected of containing the hydrolytic enzyme with a S 20 protected dihydroxyaromatic compound in which one of the hydroxy groups is protected with an enzyme-cleavable group in the presence of a heterocyclic enol phosphate compound to produce chemiluminescence and relating the chemiluminescence to the amount of the hydrolytic enzyme in the sample.

In a further aspect there is provided a method of conducting an assay of an analyte comprising reacting a hydrolytic enzyme, a protected dihydroxyaromatic compound and a heterocyclic enol phosphate compound to produce chemiluminescence, measuring the chemiluminescence and relating the chemiluminescence to the amount of the analyte, wherein the hydrolytic enzyme is provided as a label on a specific binding pair member.

-8- P:\WPDOCS\1j.spo\79928pgs.dc-09/l 1/01 These and other aspects of the invention are further described and defined in the description and claims which follow.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a plot showing the rapid generation of chemiluminescence from the reaction of acridan phosphate 1 with hydroquinone at room temperature. The reaction mixture comprised 0.66 mM acridan phosphate 1 and 4 pM hydroquinone in 2-methyl-2-amino-l-propanol (221) buffer, 0.2 M, pH 9.6 also containing 0.88 mM MgCl 2 Figure 2 is a graph relating chemiluminescence intensity to the quantity of acridan phosphate 2. Chemiluminescence emitted by reaction of 100 LL of a reagent composition consisting of varying amounts of acridan phosphate 2 (dilutions contained between 6.6 x 10 1 1 and 6.6 x 10 17 mol) in 0.2 M 221 buffer, pH 9.6, 0.88 mM MgC1, 0.1 SDS with 100 p.L of a 0.01 mM solution of 2-chlorohydroquinone (DHA- 1) in water was measured at 2.5 min.

Figure 3 is a plot showing the time profile of the chemiluminescence intensity emitted by 100 pL of a reagent consisting of a 0.33 mM solution of acridan phosphate 1 and 1 mm 4-hydroxyphenyl phosphate in 0.1 M 221 buffer, pH 9.6 triggered at 25 OC by addition of 8 x 10 17 mol. of alkaline phosphatase (AP).

-9- WO 99/40161 PCT/US98/1 489 Figure 4 is a plot showing the time profile of chemiluminescence emission as a function of the concentration of 4-hydroxyphenyl phosphate. A reagent composition consisting of a 0.33 mM solution of acridan phosphate 1 and 0.88 mM MgC12 in 0.2 M 221 buffer, pH 9.6 containing varying concentrations of 4-hydroxyphenyl phosphate (in mg/mL a, 1; b, 3.3; c, 0.66; d, 0.83; e, 10; f, 0.1; g, 0) was reacted with 8 x 10 17 mol of AP at 25 oC.

Figure 5 is a graph relating the amount of AP to chemiluminescence intensity emitted by 100 gL of a reagent composition comprising 0.66 mM acridan phosphate 1, 0.66 mM 4-hydroxyphenyl phosphate and 0.88 mM MgCl 2 in 0.2 M 221 buffer, pH 9.6. The composition (100 JL) was reacted with lL of solutions of AP containing between 8 x 10 15 and 8 x 10 20 mol of enzyme or 10 pL of water as a reagent blank at ambient temperature in the wells of a black microplate.

Light intensity was measured after 25 min. Less than 0.1 amol of AP was detected.

Figure 6 is a graph relating the amount of 9-galactosidase to chemiluminescence intensity. Solutions of 4-hydroxy phenyl 9-galactoside (1 mM) in 0.05 M phosphate buffer, pH 0.01 M NaC1, 3 mM MgCl 2 were incubated with dilutions of i-galactosidase at 37 0 C for 35 min. Portions were reacted with an equal volume of a solution of acridan phosphate 3 having the following composition: 0.2 M 221 buffer, pH 9.6, 0.88 mM MgCl 2 0.66 mM acridan phosphate 3, and 0.1 SDS. The amount of enzyme in the wells ranged from 4.9 x -15 -18 to 1.6 x 10 mol of E-galactosidase. Chemiluminescence intensity was measured at 5 min Figure 7 is a graph relating the amount of 9-glucuroni- SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 dase to chemiluminescence intensity. Solutions of 4-hydroxy phenyl Z-glucuronide (1 mM) in 0.05 M phosphate buffer, pH 0.01 M NaCI were incubated with dilutions of 9-glucuronidase at 37 0 C for 30 min. Portions were reacted with an equal volume of a solution of acridan phosphate 4 having the following composition: 0.2 M 221 buffer, pH 9.6, 0.88 mM MgC1 2 0.66 mM acridan phosphate 4, and 0.1 SDS. The amount of enzyme in the wells ranged from 3.3 x 10 14 to 3.3 x 10 mol of 9-glucuronidase. Chemiluminescence intensity was measured at 11 min.

Figure 8 is an image of a dot blot analysis of dilutions of DNA using a reagent composition containing 0.66 mM acridan phosphate 4, 0.66 mM 4-hydroxyphenyl phosphate, 0.1 SDS and 0.88 mM MgCl 2 in 0.1 M 221 buffer, pH 9.6. The image is the result of a 1 min exposure taken 3 h after contacting the membrane with detection reagent.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions: Alkyl A branched, straight chain or cyclic hydrocarbon group containing from 1-20 carbons. Lower alkyl as used herein refers to those alkyl groups containing up to 8 carbons.

Alkenyl A branched, straight chain or cyclic hydrocarbon group containing at least one C-C double bond and containing from 2-20 carbons. Lower alkenyl as used herein refers to those alkenyl groups containing up to 8 carbons.

Alkynyl A branched or straight chain hydrocarbon group containing at least one C-C triple bond and 11 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 containing from 2-20 carbons. Lower alkynyl as used herein refers to those alkynyl groups containing up to 8 carbons.

Analyte A substance the presence or amount of which is to be measured in a sample by an assay. Analytes include organic and biological molecules to which a specific binding partner having a specific binding affinity exists.

Exemplary analytes include, without limitation, single stranded or double stranded DNA, RNA, DNA-RNA complexes, oligonucleotides, antibodies, antibody fragments, antibody- DNA chimeras, antigens, haptens, proteins, lectins, avidin, streptavidin and biotin. Other exemplary analytes also include hydrolytic enzymes, inhibitors of hydrolytic enzymes and dihydroxyaromatic compounds.

Aryl An aromatic ring-containing group containing 1 to 5 carbocyclic aromatic rings, which can be substituted with 1 or more substituents other than H.

Biomedical analysis Analyses of samples of biological origin for analytes of interest. The analyses can be immunoassays, western blots, northern blots, Southern blots, DNA hybridization assays, DNA sequence analysis, colony hybridizations, gene expression analysis, high throughput drug screening, detection of infectious agents or pathogens and the like.

Dihydroxyaromatic compound An aromatic ring compound comprising an aromatic ring system, in turn comprising at least one carbocyclic ring and up to five rings, which is substituted with two hydroxy groups which are separated by an even number of ring carbon atoms. This substitution pattern is exemplified by the ortho- and para- substitution pattern on a benzene ring and by e.g. 1,4- or 2,6- 12 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 substitution pattern on a naphthalene ring. Dihydroxysubstituted biaryl ring systems such as biphenyl and binaphthyl are considered to be within the scope of the definition subject to the limitation of there being an even number of ring carbon atoms separating the two hydroxy groups. A dihydroxyaromatic compound can be substituted with additional hydroxy groups, i.e. trihydroxy, tetrahydroxy etc. and still be within the scope of the definition provided that at least one pair of hydroxy groups is separated by an even number of ring carbon atoms.

Halogen Fluorine, chlorine, bromine or iodine atoms.

Sample A fluid containing or suspected of containing one or more analytes to be assayed. Typical samples which are analyzed by the chemiluminescent reaction method are biological samples including body fluids such as blood, plasma, serum, urine, semen, saliva, cell lysates, tissue extracts and the like. Other types of samples include food samples and environmental samples such as soil or water.

Specific binding pair Two substance which exhibit a mutual binding affinity. Examples include antigen-antibody, hapten-antibody or antibody-antibody pairs, complementary oligonucleotides or polynucleotides, avidin-biotin, streptavidin-biotin, hormone-receptor, lectin-carbohydrate, IgG-protein A, nucleic acid-nucleic acid binding protein and nucleic acid-anti-nucleic acid antibody.

It should be noted that in references to "a heterocyclic enol phosphate compound" or "a dihydroxyaromatic compound" or "a protected dihydroxyaromatic compound", it is intended to include more than one of the described species unless clearly indicated 13 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 to be only the singular case.

It has been unexpectedly discovered that certain dihydroxyaromatic compounds comprising an aromatic ring system substituted with at least two hydroxy groups separated by an even number of ring carbon atoms react with a second compound which is a heterocyclic enol phosphate compound containing a heterocyclic ring group and an enol phosphate group in the presence of oxygen to generate easily detectable chemiluminescence. Exemplary aromatic ring systems embodied within the dihydroxyaromatic compounds of the present invention include benzene, biphenyl, naphthalene, anthracene, phenanthrene, pyrene, benzpyrene and naphthacene ring systems.

A preferred group of dihydroxyaromatic compounds have the formula I

OH

R

1 R4

R

2 R3

I

wherein at least one of R 1 and R 3 is an OH group, the other one of R or R and R 2 R and R are each independently selected from hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, amino, aminoalkyl, carboxyl -C(=O)OH, carboxyl ester -C(=0)OR 6 formyl alkylcarboxy OC(=O)R arylcarboxy -OC(=O)R 9 and halogen groups wherein pairs of adjacent groups, when taken together, can complete a five or six-membered aliphatic or aromatic ring and wherein R is a lower alkyl group, wherein R 7 and R 8 are each H or a lower alkyl group and wherein R 9 is an aryl ring group.

14 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 More preferred compounds of formula I are those compounds wherein R 3 is the OH group and R 1

R

2

R

4 and R are independently selected from hydrogen, alkyl, alkoxy, halogen, aryl and aralkyl groups. Still more preferably, each of R 2 R and R are hydrogen and R is selected from hydrogen, alkyl, alkoxy, halogen, aryl and aralkyl groups.

It is recognized that other dihydroxyaromatic compounds in addition to those with the two hydroxy groups substituted on the same six-membered ring are within the scope of the dihydroxyaromatic compounds and are effective in the present methods. Dihydroxyaromatic compounds containing a biaromatic ring system wherein the hydroxy groups reside on different six-membered rings are exemplary. Some particular examples of dihydroxyaromatic compounds which are useful in the methods of the present invention are listed in Table 1 for purposes of illustrating and not defining the scope of the invention.

Table 1. Dihydroxvaromatic compounds DHA-1 DHA-2 DHA-3 DHA-4 DHA-5 DHA-6 DHA-7 DHA-8 DHA-9 DHA-10 2-Chlorohydroquinone Hydroquinone 2-Phenylhydroquinone 3,3,3',3'-Tetramethyl-l,1'-spirobisindane- 5,5',6,6'-tetrol 2,3,5,6-Tetrafluorohydroquinone 2-Methylhydroquinone 2-Methoxyhydroquinone Catechol 1,2-Dihydroxyanthracene 7,8-Dihydroxy-6-methoxycoumarin SUBSTITUTE SHEET (RULE 26) WO 99/40161 WO 99/016 1PCTIUS98/1 1489 DHA-11 1-Methyl-6,7-dihydroxy-1,2,3,4tetrahydroisoquinolime DHA-12 4-Aminoresorcinol DHA-13 1, 4-Dihydroxyniaphthalene DHA-14 1, 2, 4-Trihydroxybenzene 9, 10-Bis (4-hydroxyphenyl)anthracene DHA-16 4-Bromoresorcinol DHA-17 2, 3-Dihydro-9, 10-dihydroxy-1, 4-anthraquinone DHA-18 Ellagic acid DHA-19 2, 2, 3-Dichloro-5, 8-dihydroxy-1, 4-naphthoquinone OH OH OH HO C p I I IHO OH OH OHN DHA-1 DHA-2 DHA-3 DHA-4

OH

F F :1 F F

OH

DHA- 5 OH OH OH OH CH 3 OCH 3OH .OH I N N N: OH OH DHA-6 DHA-7 DHA-8 DHA-9 CH 3

O~

HO0 0 0

OH

DHA- 13 NH DHA-12 DHA- 10 DHA- 11 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

OH

HOH

OH

DHA-14 OH 0 OH OH 0 DHA-15 0 Ho 0 oOH HO

DHA-

0

OOH

DHA-18 DHA-16 OH OH O S CHO OH

C

OH OH O ci OH OH 0 DHA-17 DHA-19 Heterocyclic enol phosphate compounds useful in the present methods and compositions for producing chemiluminescence have the formula: MnO3PO V

ZR

10

R

1 8 R1

R'

7

R

12

III

R16 R 13

R

15 R19 R 14 wherein R10 is an organic group which allows the light to be produced, wherein each of R 11

R

18 are independently selected from and wherein pairs of adjacent groups can complete a benzo-fused ring, wherein R 19 is an organic 17 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/1 1489 group containing up to 50 atoms selected from C, N, 0, S, P and halogen atoms, wherein Z is selected from 0 and S atoms, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality. In compounds of formula III where double bond isomers are possible, mixtures of the two double bond isomers can be used.

Referring back to formula III, exemplary ring structures include the structures below where the asterisk denotes the position of the exocyclic double bond. Without explicitly showing all possible substitution patterns, it is to be understood that each ring position can contain substituents other than hydrogen.

R

19

R

1 9

N

R

1 9 R 19 The group R 19 is preferably selected from alkyl, substituted alkyl, aryl, substituted aryl, aralkyl and substituted aralkyl groups. More preferred groups for R 19 include substituted and unsubstituted lower alkyl groups and substituted and unsubstituted benzyl groups.

In all of the above compounds, the groups R 11

R

18 18 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 each are independently H or a substituent group which permits the light to be produced and generally contain from 1 to 50 atoms selected from C, N, 0, S, P and halogen atoms. Representative substituent groups which can be present include, without limitation, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, alkenyl, alkynyl, alkoxy, aryloxy, halogen, amino, substituted amino, carboxyl, carboalkoxy, carboxamide, cyano, and sulfonate groups. Pairs of adjacent groups, e.g. R 1

R

12 can be joined together to form a benzo-fused ring. Specific substituents and their effects are illustrated in the specific examples below, which, however, are not to be considered limiting the scope of the invention in any way.

Each of the groups M independently are a hydrogen atom or a cationic center. A cationic center means a positively charged atom such as a sodium ion Na a group of atoms such as an ammonium ion NH or a portion of a molecule 4 with one or more sites of positive charge. Examples of the latter include dicationic compounds described in U.S.

Patent 5,451,347 to applicant and polymeric compounds with multiple cationic groups as described in applicant's U.S.

Patent 5,393,469. The positive charge on a cationic center may take any unit value, i.e. 1, 2, 3 etc. Exemplary cationic centers include, without limitation, alkali metal ions, alkaline earth ions, quaternary ammonium ions and quaternary phosphonium ions and are present in the number required by their valence. If two groups are required to be present in the compound of formula I for 19 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 electroneutrality, they may be the same or different.

Preferred counter ions are the alkali metal ions. More preferred are sodium and potassium ions.

The organic group R 10 can be any group which allows light production. This means that the presence of any group as the R1 0 group does not prevent the ability of the compound of formula III to ultimately produce chemiluminescence. By the latter is meant that when a compound of formula III is reacted with the dihydroxyaromatic compound in the presence of oxygen, chemiluminescence is produced immediately and may involve the production of one or more chemiluminescent intermediates.

The group R 10 preferably contains from 1 to 50 atoms selected from C, N, O, S, P and halogen atoms exclusive of the necessary number of H atoms required satisfy the valencies of the atoms in the group. Groups which can function as the R 10 group include, without limitation, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl and substituted aralkyl groups. Substituent groups other than H atoms, such as ionic groups or polar groups, can be incorporated in various numbers and at selected positions on the carbon chain or ring of R 10 in order to modify the properties of the compound or to provide for convenience of synthesis of the final phosphate compound. Such properties include, for example, chemiluminescence quantum yield, rate of reaction with the enzyme, maximum intensity of light emission, duration of light emission, wavelength SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 of light emission and solubility in the reaction medium.

Specific substituents and their effects are illustrated in the specific examples below, which, however, are not to be considered limiting the scope of the invention in any way.

A preferred class of compounds has the formula IIIa below wherein Z is an O or S atom, wherein R 19 is a lower alkyl group and wherein Ar is an aryl ring group containing at least one carbocyclic aromatic ring and which can be further substituted.

3 P-O /Z-Ar

R

18 i R 11 R17 1 R12 R1 N R13

R

1 5

R

19 R4 IIIa The groups R to R 18 which may be the same or different, each are a substituent which may contain from 1 to atoms selected from C, H, N, O, S, P and halogen atoms and which permit the light to be produced and may include, without limitation, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl such as benzyl, alkenyl, alkynyl, alkoxy, aryloxy, halogen, amino, substituted amino groups, carboxyl, carboalkoxy, carboxamide, cyano, and sulfonate groups. One preferred set of compounds has the groups R 11 to R 18 selected from hydrogen and lower alkoxy groups such as methoxy, ethoxy, t-butoxy and the like. Other preferred compounds have the formula IIIb where Z is 0 or S and Ar is selected from a phenyl group, a substituted phenyl group or a naphthyl group.

21 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 Na 2 0 3 PO ZAr IIIb I IIIb CH3 Another preferred set of heterocyclic enol phosphate compounds contain a group -A-Q which permits the compound to be used as a label. Preferred labeling compounds contain the group -A-Q as a substituent on R 10 or R 19 or at any one of the R 11

R

1 8 positions. Examples of heterocyclic enol phosphate labeling compounds have formulas IIIc, d or e.

3 PO. ZAr-A-Q Na20 3 PO ZAr R 8 R2 R 17

R

12 8 1 R16 N R 1 3

R

1 6 N R

R

15 R19 R 14

R

1 5 19 R 14

A-Q

IIIc IIId Na 2 0 3 PO ZAr R 18

R

11

R

1 7 R1 -A-Q IIIe In the group A is a spacer group selected from C -C 1 0 alkylene, and C2-C0 oxyalkylene groups and Q is a linking group capable of forming a covalent bond with a molecule to be conjugated such as an analyte or a specific binding partner. Linker groups Q can be electrophilic 22 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 moieties such as halogen, diazo, -NCO, -NCS, -CHO, acid anhydride, oxiranyl, succinimidoxycarbonyl, maleimide, cyano, triazole and tetrazole groups and the like as are generally known in the art. Alternatively, linker groups Q can be nucleophilic moieties such as hydroxyl, -COOH, thiol, primary amino, secondary amino groups.In a compound of formula IIIe it is intended that the group A-Q can be present on either one of the two benzene rings.

The following specific compounds are particularly preferred for use in the presently described novel chemiluminescent methods: Na 2 03PO .OPh Na 2 0 3 PO, SPh N

N

CH

3 CH 3 1 2 Na 2 0 3 PO.. S-C Na 2 0 3 PO.. S

N

I 3 CH3

CH

3 4 Reaction of a compound of formula III with a dihydroxyaromatic compound in an aqueous buffer solution produces bright chemiluminescence which rapidly reaches maximum intensity at room temperature.

23 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/1 489

M

2 0 3 PO ~ZR 10

R

18

R

1

R

1 8 0 Rll

R

1 7

R

12 Dihydroxyaromatic

R

1 compound, I I R1 6 N R 3 compound, 2 R16 N R 13

R

1 5 R19 R 14

R

15 R1 9 R14 III IV Light While we do not wish to put forth a specific mechanistic explanation for this discovery at this point, it is believed that chemiluminescence is emitted from the electronically excited state of a ketone compound IV formally derived from oxidation of the double bond of the heterocyclic enol phosphate compound. A necessary condition for the production of chemiluminescence is that the reaction produces sufficient energy to form the excited state of IV and that IV is fluorescent. The active oxidant may be oxygen, a reduced form of oxygen such as superoxide ion, peroxide ion or a peroxy radical, or the active oxidant may be an oxidized product of the dihydroxyaromatic compound, such as the quinone or semiquinone form.

Chemiluminescence emission from the present reactions and methods is typically produced as a 100-200 nm wide band of emission and exhibits a maximum intensity at wavelengths in the near ultraviolet to the visible region of the electromagnetic spectrum. Typical wavelengths of maximum intensity, X are in the range of 350-500 nm. It is contemplated that phosphate compounds of formula III 24 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/1 1489 bearing a covalently linked fluorophore could undergo intramolecular energy transfer resulting in emission at longer wavelengths from the excited state of the fluorophore.

Chemiluminescent light emitted by the present method can be detected visually or by any other suitable known means such as with a luminometer, x-ray film, high speed photographic film, a CCD camera, a scintillation counter or a chemical actinometer. Each detection means has a different spectral sensitivity. The human eye is optimally sensitive to green light, CCD cameras display maximum sensitivity to red light, x-ray films with maximum response to either UV, blue light or green light are available. Choice of the detection device will be governed by the application and considerations of cost, convenience, and whether creation of a permanent record is required.

The chemiluminescent reactions of the present invention comprising the reaction of compounds of formula I and III in the presence of oxygen may find use as chemical light sources, an example of which is the familiar light stick and related novelty items or for emergency lighting. Another use is in methods of detecting a compound of formula I in a sample in biomedical analysis, food analysis or environmental analysis of pollutants.

The chemiluminescent reactions of the present invention can also be used in a method of conducting an SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/ 1489 assay of an analyte in a sample by means of detectably labeled specific binding pairs. In one embodiment of such an assay method, either compound I or compound III is provided as a label compound. The label compound contains a substituent which bears a reactive labeling group, whereby the reactive group is capable of forming a covalent attachment to one member of a specific binding pair to form a detectably labeled specific binding pair member. The assay method further comprises reacting the labeled specific binding pair member and its partner for detecting the analyte to form a labeled specific binding pair and reacting the labeled specific binding pair with either compound I or compound III, as required, to produce chemiluminescence for detecting the analyte. Preferably the heterocyclic enol phosphate is provided as the label.

In another embodiment of an assay method, either compound I or compound III is provided as a detectable species encapsulated in a liposome which is conjugated to a specific binding pair member. Methods for preparing liposomes containing detectable species and their use as detectable labels in chemiluminescent assays are described in U.S. Patent No. 5,595,875, which is incorporated herein by reference.

In another embodiment of an assay method, both compound I and compound III are each provided encapsulated in a separate latex particulate material to which are affixed complementary specific binding pair members.

Methods for preparing latex particulate materials 26 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 containing detectable species and their use as detectable labels in chemiluminescent assays are described in U.S.

Patent No. 5,578,498, which is incorporated herein by reference.

When measuring chemiluminescence in an assay procedure, the measurement can comprise measuring the light intensity at a fixed point in time relative to the event in the assay procedure, or at a series of time points or measuring the maximum instantaneous intensity or measuring the total quantity of light or the period of time until a specified light intensity is reached.

In practicing the methods of the present invention, oxygen is typically supplied dissolved in a solution of the reactants at equilibrium with the atmosphere. The atmosphere above the solution can be enriched or replaced with oxygen in order to increase the concentration of dissolved oxygen. The solution can also be placed in a sealed vessel capable of being pressurized and the gas space in the vessel pressurized with oxygen. Other modes of delivering oxygen, for example, sparging air or oxygen or generating oxygen by a chemical reaction are considered to fall within the scope of the methods described and claimed herein.

In another aspect, the present invention relates to reagent compositions for producing chemiluminescence comprising an aqueous buffer with a pH in the range of 7- 10.5, a compound of formula I at a concentration of 0.001mM and a compound of formula III at a concentration of 27 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 0.001-20 mM. Preferably the pH is in the range of 8-10 and more preferably the pH is in the range of 9-10.

In a preferred embodiment the composition additionally comprises an anionic surfactant at a concentration preferably between 0.001 and 5 mg/mL in the final reaction solution, more preferably between 0.01 and 2.5 mg/mL.

A preferred reagent composition for producing chemiluminescence comprises an aqueous buffer with a pH in the range of 8-10, a dihydroxyaromatic compound of formula I at a concentration of about 0.05-5 mM, an acridan phosphate of formula IIIc at a concentration of about 0.05-5 mM and optionally, an anionic surfactant at a concentration preferably between 0.01 and 2.5 mg/mL. In a more preferred composition, the dihydroxyaromatic compound of formula I is selected from hydroquinone, 2chlorohydroquinone, 2-phenylhydroquinone and 2,3,5,6tetrafluorohydroquinone.

Chemiluminescent Detection of Hydrolvtic Enzymes The present chemiluminescent reaction can serve as the basis for an enzymatic chemiluminescent reaction wherein a dihydroxyaromatic compound is produced by the reaction of a hydrolytic enzyme with a protected dihydroxyaromatic compound in which one of the hydroxy groups is protected with an enzyme-cleavable group to remove the enzymecleavable group. The dihydroxyaromatic compound undergoes reaction with a heterocyclic enol phosphate compound in the presence of oxygen to generate chemiluminescence.

28 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 Preferred protected dihydroxyaromatic compound have formula II wherein one of the hydroxy groups of a dihydroxyaromatic compound, I, is protected with a group cleavable by a hydrolytic enzyme. Protected dihydroxyaromatic compounds useful in these methods comprise compounds of formula II:

OH

R 1 R R 2

R

3

II

wherein one of R or R is an OX group wherein X is a group which is removable by a hydrolytic enzyme, the other one of R 1 or R and R 2 R and R 5 are each independently selected from hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, amino, aminoalkyl, carboxyl -C(=O)OH, carboxyl ester -C(=0)OR 6 formyl alkylcarboxy OC(=O)R arylcarboxy -OC(=O)R and halogen groups wherein pairs of adjacent groups, when taken together, can complete a five or six-membered aliphatic or aromatic ring and wherein R 6 is a lower alkyl group, wherein R 7 and R 8 are each H or a lower alkyl group and wherein R 9 is an aryl ring group.

The X group in the protected dihydroxy aromatic compounds of the present invention can be any group which can be cleaved by a hydrolytic enzyme. Hydrolytic enzymes are those enzymes classified in Class E.C. 3.1 or 3.2 of the enzyme classification system and include broadly phosphatase, phosphodiesterase, nuclease, glycosidase, 29 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 lipase and esterase enzymes. Specific examples include acid phosphatase, alkaline phosphatase, phosphodiesterase, phospholipase, Z-D-galactosidase, Z-glucuronidase, 9-glucosidase, lactase, carboxyl esterase, and acetylcholinesterase. Possible O-X groups include any chemical leaving group which is stable under the conditions of use and can be cleaved by reaction with an enzyme, including without limitation alkyl or aryl carboxyl ester, inorganic oxyacid salt including phosphate and sulfate, and oxygen-pyranoside including a- and S-D-galactoside, a- and 9-glucuronide, and a- and 8-glucoside groups and the like as are apparent to those of ordinary skill in the art. Preferred hydrolytic enzymes are alkaline phosphatase, acid phosphatase, 9-D-galactosidase, S-glucuronidase and Z-glucosidase.

Formation of the dihydroxyaromatic compound by reaction of a compound of formula II with a hydrolytic enzyme can be performed in the presence or absence of the heterocyclic enol phosphate compound (III) so that the formation of I and the generation of chemiluminescence can proceed concurrently or consecutively.

In one method of producing chemiluminescence, the protected dihydroxyaromatic compound is reacted with an appropriate hydrolytic enzyme in the presence of a heterocyclic enol phosphate compound in a buffer. The pH is maintained at a value which is conducive to enzyme activity and the chemiluminescent reaction usually between about pH 8 and 10 and preferably between about pH 9 and SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCTIUS98/11489 Conducting the reaction in this manner results in a continuous chemiluminescence signal.

In an alternate mode of performing the present chemiluminescent reactions, the protected dihydroxyaromatic compound is reacted with a hydrolytic enzyme in a buffer or on a solid surface at a first pH, preferably in the range 5.0 9.5, for a first period of time, preferably ranging from a few seconds to several min. Then this solution or a measured portion thereof is reacted with a second solution containing the heterocyclic enol phosphate compound and optionally an anionic surfactant, and the light measured either by measuring the peak intensity, or integrating for a fixed time period or until light emission has ceased. The pH of the second -solution should be 8 and preferably between 9 and 10 and will typically be a buffer solution.

This mode of generating the chemiluminescence may be advantageous in assays in which a large number of samples is processed simultaneously for measurement at a later time. This two-step mode is also useful for measuring hydrolytic enzymes with optimum activity at acidic to neutral pH such as 9-galactosidase or acid phosphatase. An optional step which can also be incorporated into such a chemiluminescent reaction or assay is to add an enzyme inhibitor to the reaction system after the first period of time to stop all further enzyme action.

It will be appreciated that the selection between these 31 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 two reaction modes will be governed by various factors and the particular use or application. Nevertheless both modes are effective. For enzymes with an acidic pH optimum it may be advantageous to carry out the reaction in two discrete steps, each at an optimum pH, temperature, ionic strength, buffer salt, etc.

In producing chemiluminescence from the reaction of compound II with a hydrolytic enzyme in the presence of a heterocyclic enol phosphate, the reaction is generally performed at a temperature between 5 OC and 50 oC, preferably between 20 OC and 40 °C in an aqueous buffer solution at a pH between 7 and 10.5, preferably between and 10. Compound III is used at a concentration between 1 pM and 20 mM, preferably between 50 M and 5 mM.

Compound II is used at a concentration between 1 p.M and mM, preferably between 50 pM and 5 mM.

An anionic surfactant can also be employed in the chemiluminescent reaction of the present invention.

Anionic surfactants function decreasing background chemiluminescence from the heterocyclic enol phosphate in the absence of the dihydroxyaromatic compound. When used, anionic surfactants will be employed in an amount between 0.01 and 5 mg/mL in the final reaction solution, more preferably between 0.1 and 2.5 mg/mL. Anionic surfactant enhancers include alkyl sulfates and alkyl sulfonates.

More extensive lists of exemplary structures of each category of surfactant can be found in any standard treatise on surfactants. Preferred surfactants are C -C 10 32 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/I1489 alkyl sulfates such as sodium dodecyl sulfate, SDS.

An important use of the enzymatic chemiluminescent methods is for detecting the presence or amount of an analyte in an assay procedure by a chemiluminescent reaction. The method comprises the steps of contacting a sample suspected of containing the analyte with a chemiluminescent compound of the present invention and a hydrolytic enzyme, detecting the light produced in a qualitative method and, if quantitation is desired, relating the amount of light produced to the amount of the analyte. The relationship between light intensity and amount of analyte can be easily discerned by constructing a calibration curve with known amounts of the analyte. The chemiluminescent compound is typically used in a concentration of about 10 5 M to about 10 2 M, preferably between about 10 4 M and about 10 3 M. The hydrolytic enzyme is preferably below about 10 9 M when detected in a solution.

There are several categories of analytes which can be assayed by the present methods. These include hydrolytic enzymes, in which case it would be unnecessary to add additional hydrolytic enzyme. For example, when the hydrolytic enzyme is alkaline phosphatase, such a determination may find use e.g. in measuring the level of alkaline phosphatase in blood serum as an indication of the status of a patient's liver function or as an index of certain disease conditions. Measurement of prostatic acid phosphatase is also useful as a clinical diagnostic index 33 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 of prostate cancer.

Detection and measurement of enzyme activity by a chemiluminescent assay of the present invention will find use in gene expression assays. 9-Galactosidase, fglucuronidase and alkaline phosphatase, in particular an isozyme produced in the placenta which is excreted, are useful as reporter genes Alam, J. Cook, Anal. Biochem.

188, 245-54 (1990)). In this type of assay, a gene responsible for expression of a reporter enzyme is cloned into the genetic material of an organism via a plasmid in the vicinity of a promoter or enhancer sequence. The effect of the promoter or enhancer sequence on transcriptional activity is gauged by measuring the level of production of reporter enzyme.

Analyte used in this context also includes inhibitors of hydrolytic enzymes. Inhibitors include compounds which act reversibly by competing with a second substrate such as a protected dihydroxyaromatic compound as well as those inhibitors which act irreversibly by deactivating the enzyme. For example, inhibitors of alkaline phosphatase include inorganic phosphate, levamisole, tetramisole and other imidazo[l,2-b]thiazoles, L-phenylalanine and Lhomoarginine and are identified in R.B. McComb, G.N.

Bowers, S. Posen in Alkaline PhosDhatase, Plenum Press, New York 1979, pp. 268-275, 332-334, 394-397, 410-413.

Inhibitors of other hydrolytic enzymes are generally known to one of skill in the art of enzyme assays. When using the chemiluminescent reaction of the present invention for 34 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 detecting an enzyme inhibitor, the appropriate hydrolytic enzyme is reacted with a compound of formula II to form a dihydroxyaromatic compound I which is reacted with a heterocyclic enol phosphate III in the presence and absence of the inhibitor substance and the results are compared to determine the presence or amount of the inhibitor. The difference in the chemiluminescence emitted is indicative of the presence of the inhibitor.

Analyte used in this context further includes various classes of organic and biological molecules which can be labeled with a hydrolytic enzyme or can be specifically detected through enzyme-labeled specific binding partners.

The enzyme can be incorporated directly as a label on the analyte binding compound. Alternately the analyte binding compound may be bound to at least one enzyme-labeled specific binding substance for the analyte.binding compound. Exemplary of this format would be a nucleic acid hybridization assay using an enzyme-labeled detection oligonucleotide. Alternately the analyte binding compound can be labeled with at least one second specific binding substance, e.g. biotin, which is then bound to a enzymelabeled binding partner for the second specific binding substance, e.g. avidin.

Biological molecules which can be conjugated to one or more molecules of a hydrolytic enzyme include DNA, RNA, oligonucleotides, antibodies, antibody fragments, antibody-DNA chimeras, antigens, haptens, proteins, lectins, avidin, streptavidin and biotin. Complexes SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCTUS98/1 1489 including or incorporating hydrolytic enzymes such as liposomes, micelles, vesicles and polymers which are functionalized for attachment to biological molecules can also be used in the methods of the present invention.

Techniques for performing enzyme assays are well known.

With the guidance provided by the examples as taught herein, variations of procedures for preparing samples, determining appropriate quantities and ratios of reagents, reaction times, constructing calibration curves and the like will be within the ability of one of ordinary skill in the art to devise as a matter of routine experimentation.

Since the reaction is catalyzed by the hydrolytic enzyme, exceedingly small quantities of the enzyme are sufficient to produce a detectable amount of light.

Sensitivities of 1 amol (1 x 10 18 mol) have been achieved. The ability to detect such small amounts of hydrolytic enzymes make the present chemiluminescent technology suitable for analyses of many types of analytes using enzyme-linked assays. Such analyses and assays require the ability to detect small quantities of hydrolytic enzymes due to low abundance of the analyte in the sample to be analyzed or to limited sample quantity.

In this type of assay, alkaline phosphatase is conjugated to one member of a specific binding pair. An example is chemiluminescent enzyme-linked immunoassays, such as the so-called enzyme-linked immunosorbent assay or ELISA. Such assays are commonly used in manual format as well as on 36 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 automated multi-test immunoassay systems. In a typical immunoassay, the analyte hapten, antigen or antibody is assayed by detecting the presence or amount of an enzymelabeled specific binding partner for the analyte or an enzyme-labeled analog of the analyte. Various assay formats and the protocols for performing the immunochemical steps are well known in the art. These assays fall broadly into two categories. Competitive assays feature an immunological binding of a specific antibody with the analyte and an analyte analog, e.g. a detectably labeled analyte molecule. Sandwich assays result by the sequential or simultaneous binding of two antibodies, one of which is detectably labeled, with the analyte. The detectable enzyme-labeled binding pair so formed can be assayed with the compounds and methods of the present invention. Measurement can be performed with .enzyme-labeled species attached to a solid surface or support including beads, tubes, microwells, magnetic particles, test strips, membranes and filters such as are in common use in the art. The detectable enzyme-labeled species can also be present free in solution or enclosed within an organized assembly such as a liposome in which case a lytic agent is employed to lyse the liposome and free the detectable enzyme.

Another exemplary use is the detection of proteins by the technique of Western blotting. A sample containing a protein of interest as the analyte is subject to electrophoretic separation. The separated proteins are 37 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 transferred to a blotting membrane by capillary action or with the aid of an electric field. Such transferred protein is typically detected with a specific primary antibody and an enzyme-labeled secondary antibody which recognizes and binds to the primary antibody.

Visualization of marker enzyme activity reflects the presence of the analyte protein. To adapt the methods of the present invention for Western blotting, a secondary antibody conjugated to a hydrolytic enzyme such as alkaline phosphatase or E-galactosidase can be employed and enzyme activity measured with chemiluminescence using a compound of formula II as the enzyme substrate and a compound of formula III as the chemiluminescent reagent.

Variations on this technique such as using biotinylated antibodies and avidin-enzyme conjugates are considered within the scope of assays able to be performed using the inventive methods.

In addition to the aforementioned antigen-antibody, hapten-antibody or antibody-antibody pairs, specific binding pairs also can include complementary oligonucleotides or polynucleotides, avidin-biotin, streptavidin-biotin, hormone-receptor, lectincarbohydrate, IgG-protein A, nucleic acid-nucleic acid binding protein and nucleic acid-anti-nucleic acid antibody.

A particularly useful application of the present detection methods is the detection of nucleic acids by the use of enzyme-labeled nucleic acid probes. Methods for 38 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 analysis and chemiluminescent detection of nucleic acids using enzyme-labels, for example, solution hybridization assays, DNA detection in Southern blotting, RNA by Northern blotting, DNA sequencing, DNA fingerprinting, colony hybridizations and plaque lifts are all well established techniques. The enzyme label AP or 9gal) can be present as a direct conjugate with a probe oligonucleotide or capture oligonucleotide or it can be incorporated through indirect linking means using artknown methods. Examples of indirect linking means include using hapten-labeled oligonucleotides and anti-haptenenzyme conjugates or biotinylated oligonucleotides and avidin-enzyme conjugates. Such nucleic acid assays can be performed on a blotting membrane or in solution using oligonucleotides attached to solid surfaces including beads, tubes, microwells, magnetic particles, test strips such as are known in the art.

In another aspect, the present invention relates to a reagent composition for producing chemiluminescence by reaction with a hydrolytic enzyme comprising a protected dihydroxyaromatic compound of formula II to produce the dihydroxyaromatic compound of formula I which in the presence of a compound of formula III and oxygen produces chemiluminescence. The reagent composition comprises an aqueous buffer with a pH in the range 7-10.5, a compound of formula II at a concentration of 0.001-20 mM and a compound of formula III at a concentration of 0.001-20 mM.

Formulations for chemiluminescent reaction with a 39 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US981 1489 hydrolytic enzyme can further comprise a metal salt such as a magnesium or zinc at a concentration of 0.01-10 mM for increasing or sustaining the activity of the enzyme.

In a preferred embodiment the composition additionally comprises an anionic surfactant at a concentration preferably between 0.01 and 5 mg/mL in the final reaction solution, more preferably between 0.1 and 2.5 mg/mL.

A preferred reagent composition for producing chemiluminescence in a one-step process by reaction with a hydrolytic enzyme comprises an aqueous buffer with a pH in the range of 8-10, a protected dihydroxyaromatic compound of formula II at a concentration of about 0.05-5 mM, an acridan phosphate of formula IIIc at a concentration of about 0.05-5 mM and optionally, an anionic surfactant at a concentration preferably between 0.1 and 2.5 mg/mL. In a more preferred composition, the protected dihydroxyaromatic compound of formula II has the formula:

OH

Ox

II

wherein X is the group removable by the hydrolytic enzyme.

In order to more fully describe the various aspects of the present invention, the following examples describing compounds, methods of preparation, compositions and methods of use are presented. The examples are to be considered illustrative and do not limit the scope of the invention.

SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

EXAMPLES

1. Synthesis of Acridan Derivative 1.

Na 2 0 3 PO OPh

NNN

N

CH

3 1 a. Phenvl acridine-9-carboxvlate. Acridine-9-carboxylic acid (1 g, 4.1 mmol) was suspended in thionyl chloride mL) and the reaction mixture was refluxed for 3 h. The solvent was removed under reduced pressure leaving a yellow solid which was dissolved in CH C 2 and pyridine (350 jL) under argon. This solution was cooled in an ice bath and a solution of phenol (0.78 g, 8.2 mmol) in CH 2 Cl2 was added dropwise. The reaction mixture was stirred overnight at room temperature. After evaporation of solvent, the residue was redissolved in ethyl acetate and washed with water. The organic layer was dried over MgSO 4 and concentrated to obtain a crude material which was chromatographed on silica gel (30 ethyl acetate/hexane) to yield the pure product as a yellow solid. H NMR (CDC1 3 6 7.35-7.57 5H), 7.63-8.37 8H).

b. Phenvl 10-methvlacridinium-9-carboxvlate trifluoromethanesulfonate. Phenyl acridine-9-carboxylate (530 mg, 1.7 mmol) was dissolved in CH C 2 (5 mL) under argon and methyl trifluoromethanesulfonate (1 mL, 8.8 mmol) was 41 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 added. The solution was stirred overnight at room temperature to yield a thick yellow precipitate. This precipitate was filtered, washed with ether and dried to obtain the product as yellow crystals. 1 H NMR (acetone-d 6 5.22 3H), 7.47-7.71 5H), 8.23-9.07 8H).

c. Phenvl 10-methvlacridan-9-carboxvlate. Phenyl 10-methylacridinium-9-carboxylate trifluoromethanesulfonate (10 mg, 0.0216 mmol) was suspended in absolute ethanol (10 mL) and the mixture was refluxed for 15 min to obtain a clear solution. Ammonium chloride (88 mg, 1.6 mmol) was added by portions to the solution followed by zinc (108 mg, 1.6 mmol). Addition of zinc caused the yellow color of the solution to disappear immediately. The colorless solution was refluxed for 2 h.

TLC of the reaction mixture showed complete conversion to a non polar material. The solution was filtered and precipitate was washed with ethanol (3 x 20 mL). The filtrate was concentrated to obtain an off-white solid which was redissolved in CH Cl and washed with water (2 x 2 2 mL). The organic layer was dried over Na SO and 2 4 concentrated to yield the crude product which was purified by preparative TLC using (30% ethyl acetate:hexane). Pure product was obtained as an off-white solid. H NMR (CDC1 3 6 3.38 3H), 5.16 1H), 6.89-7.37 13H); 13 C NMR (CDC1 6 33.29, 49.72, 112.93, 120.19, 121.36, 125.73, 128.67, 129.16, 129.26, 142.37, 151.04, 170.22.

42 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 d. 9-(PhenoxvyhosDhorvloxvmethylidene)-10-methylacridan, bis(cvanoethvl) ester. A three-neck flask was purged with argon and charged with 5 mL of anhydrous THF and diisopropylamine (0.04 mL, 0.29 mmol). The flask was cooled in a dry ice-acetone bath. To this solution was added n-butyl lithium (0.116 mL, 0.29 mmol). After 20 min, a solution of the acridan ester from step (c)(70 mg, 0.22 mmol) in 5 mL of THF was added to this solution and stirring was continued for 30 min at -78 oC. Finally a solution of POC1 3 (0.027 mL, 0.29 mmol) and pyridine (0.023 mL, 0.29 mmol) in 3 mL of THF was added and the dry ice bath was removed. After 45 min, pyridine (0.039 mL, 0.58 mmol) and 3-hydroxypropionitrile (0.094 mL, 1.16 mmol) was added and stirring maintained over night. Then it was filtered and solvent was removed from the filtrate.

The residue was subjected to prep. TLC (80% ethyl acetate/ hexane) to give the pure product; H NMR (CDC1 6 2.35- 2.54 4H), 3.47 3H), 3.79-3.90 2H), 3.98-4.08 13 2H), 6.825-7.45 12H), 7.80-7.83 (dd, 1H); C NMR (CDC1 3 6 19.12, 19.24, 33.63, 62.19, 62.49, 88.72, 92.82, 112.40, 112.52, 115.83, 116.07, 119.68, 120.44, 120.71, 123.81, 126.69, 128.03, 128.27, 128.58, 130.09, 142.39, 143.06, 165.73, 202.09.

e. 9-(Phenoxvyhosphorvloxvmethvlidene)-10-methylacridan. disodium salt A solution of the bis(cyanoethyl) phosphate compound (2.897 g, 5.77 mmol) in mL of acetone was purged with Ar for 30 min. An Ar- 43 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 purged solution of 479 mg (12 mmol) of NaOH in 7.5 mL of water was added dropwise and the solution stirred over night. The precipitate which had formed was filtered, washed with 50 mL of Ar-purged acetone and air-dried. The yield was 3.473 g of 1 as a white solid which contained some water. 1H NMR (D20) 6 3.326 3H), 6.825- 7.45 (m, 13H), 7.80-7.83 1H); 3C NMR (D20) 6 32.95, 102.86, 102.92, 112.30, 115.85, 120.68, 121.01, 122.35, 122.41, 122.62, 127.48, 127.66, 128.23, 129.66, 143.17, 143.32, 144.66, 156.01; 31p NMR (D20) 6 0.581 (rel. to ext. H PO 2 3 4 2. Synthesis of Acridan Derivative 2.

Na 2 03PO. SPh

N

I

CH

3 2 a. 9-(Phenvthiophosphorvloxvmethylidene)-10-methylacridan, bis(cvanoethvl) ester. Phenyl 9-thiocarboxylate prepared as decribed in applicant's PCT application W095/28495 (70 mg, 0.2 mmol) was added to a solution of LDA (0.24 mmol) in THF. After stirring 30 min at -78 C, a solution of POC1 (25 gL) and pyridine (21 gL) in 4 mL of THF was added. The dry ice bath was removed and stirring continued for 30 min. TLC (30% ethyl acetate/ hexane) showed that the starting material was completely reacted. The solution was cooled in an ice bath and 44 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 treated with pyridine (210 L) and 3-hydroxypropionitrile (44 gL) in 4 mL of THF. After stirring over night at room temperature, the precipitated pyridine-HCl was filtered away and the reaction solvent evaporated in vacuo. The residue was subjected to prep. TLC (ethyl acetate) from which the product was isolated; 'H NMR (CDC13) 6 2.4-2.6 4H), 3.521 3H), 3.8-4 2H), 4.0-4.1 2H), 6.9-7.2 4H), 7.22-7.5 7H), 7.75-8 (dd, 2H).

b. 9-(Phenvlthiophohohorvloxvmethvlidene)-10-methvlacridan, disodium salt A solution of the bis(cyanoethyl) phosphate compound (0.59 g, 1.21 mmol) in mL of acetone was purged with argon for 30 min. An Arpurged solution of 104 mg (2.6 mmol) of NaOH in 10 mL of water was added dropwise and the solution stirred under argon over night. The precipitate which had formed was suction filtered, washed with 50 mL of Ar-purged acetone and air-dried. The yield was 0.50 g of 5 as a slightly yellow solid which contained a slight amount of acetone.

1 H NMR (DO2) 3.36 3H), 6.9-7.4 11H), 7.75-7.8 (d, 1H), 8.2-8.22 1H) 3 1 P NMR (D20) 6 1.85.

SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 Example 3. Synthesis of Acridan Derivative 3.

Na 2 0 3 PO.S -0

N

CH

3 3 a. 4'-Chlorophenyl acridine-9-thiocarboxlate. 4- Chloro-thiophenol (4.75 g) and 7.22 g of acridine-9carbonyl chloride were dissolved in 100 mL of CH Cl followed by 12.1 mL of pyridine. The reaction mixture became orange- brown. The mixture was stirred overnight at room temperature under argon. After evaporation of solvent, the solids were washed with 100 mL of hexanes, filtered, washed with another 100 mL of hexanes, filtered, and then with a 500 mL of water, filtered and airdried.The thioester was obtained (8.71 g) as a slightly brownish yellow solid. H NMR (CDC1 6 7.47-7.50 2H), 7.58-7.67 4H), 7.81-7.86 2H), 8.12 2H), 8.29 2H).

b. 4'-Chlorophenvl acridan-9-thiocarboxvlate. 4'- Chlorophenyl acridine-9-thiocarboxylate (2.0 g) was dissolved in CH2C 2 (25 mL). The solution was purged with argon and then 3.72 g of zinc powder added followed by 0.45 mL of acetic acid. TLC showed the starting material was consumed in 20 min. The mixture was filtered and the solids washed with CH Cl The combined CH Cl solutions 46 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 were washed with water (3 x 50 mL) and dried. A small amount of the product was purified by prep. TLC for analytical characterization. The remainder of the product was used without further purification. H NMR (CDC1 6 5.22 1H), 6.28 1H), 6.79-7.31 12H).

c. 4'-Chlorophenvl 10-methvlacridan-9-thiocarboxvlate. 4'- Chlorophenyl acridan-9-thiocarboxylate (2.0 g) was dissolved in CH 2 Cl2 (30 mL) under argon and methyl triflate (6.5 g) was added and stirred over night. The brown solution was evaporated and the crude product purified by column chromatography using 30 ethyl acetate/hexanes.

The pure product (1.8 g) was thereby obtained. H NMR (CDC1 3 8 3.47 3H), 5.09 1H), 7.02-7.39 12H).

d. 9-(4-Chlorophenvlthiophosphoryloxvmethvlidene)bis(cyanoethvl) ester. 4-Chlorophenyl 10-methylacridan-9-thiocarboxylate (0.70 g) in 10 mL of anhydrous THF was added dropwise to a solution of LDA (1.4 eq.) in THF at -78 C. After stirring 60 min at -78 C, the yellow solution was treated with a solution of POC1 (0.517 g) and pyridine (1.52 mL) in 4 mL of THF slowly.

The dry ice bath was removed after 30 min and stirring continued for 1 h. The solution became yellow and formed a precipitate.

The mixture was cooled in an ice bath and treated with 3-hydroxypropionitrile (0.89 g) and 1.0 mL of pyridine.

The ice bath removed after the addition was complete.

47 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 After stirring over night at room temperature, the precipitated pyridine-HCl was filtered off and washed with THF. The combined filtrates were evaporated in vacuo and the brown material obtained was dissolved in ethyl acetate and washed with 4 x 25 mL of water. The ethyl acetate solution was dried and concentrated. The residue was separated by column chromatography using 80-100 ethyl acetate/hexanes from which the 0.325 g of the product was isolated; H NMR (CDC1 6 2.48-2.64 4H), 3.53 3H), 3.86-4.16 4H), 6.94-7.94 12H); 3 1 P NMR (D2) 6 9.48 e. 9-(4-Chlorophenvlthiophosphorvloxvmethvlidene)disodium salt A solution of the bis(cyanoethyl) phosphate compound (0.325 g) in 10 mL of acetone was purged with argon. A solution of,2.5 M NaOH (473 gL) was added followed by an additional 500 LL of water. The solution was stirred under argon over night.

The precipitate which had formed was suction filtered and air-dried. The mother liquor was found to contain the mono(cyanoethyl)-protected compound (140 mg). A second crop of the disodium salt was obtained by repeating the NaOH deprotection. 'H NMR (DO2) 6 3.35 3H), 6.92-7.36 10H), 7.78 1H), 8.20 1H) 31 NMR (D20) 6 1.22 48 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 Example 4. Synthesis of Acridan Derivative 4.

3 PO S I I

N

I

CH

3 4 a. Naphthvl acridine-9-thiocarboxvlate. 2-Naphthalenethiol (48.81 g) and the acridine-9-carbonyl chloride prepared from 65.17 g of acridine-9-carboxylic acid were dissolved in 100 mL of CH 2 C 2 followed by the addition of 120 mL of pyridine. The mixture was stirred overnight at ro6m temperature under argon. After evaporation of solvent, the solids were washed with 500 mL of hexanes, filtered, washed with another 500 mL of hexanes, filtered, and then washed with 600 mL of water, filtered and airdried overnight. The thioester was dissolved in 1500 ml of CH C1 dried over sodium sulfate, filtered, and dried in vacuo. The thioester was obtained (94.67 g) as a brown solid. 1H NMR (CDC1 6 7.54-8.00 11H), 8.17-8.31 (m, 4H).

b. Naphthyl 10-methvlacridinium-9-thiocarboxvlate triflate. Naphthyl acridine-9-thiocarboxylate (26.38 g) was suspended in CH 2 C12 (200 mL). Methyl trifluoromethanesulfonate (24.5 ml) was added and the mixture left to stir overnight. The mixture was filtered 49 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 and the solids washed with CH 2 Cl2 (300 ml) and hexanes (500 ml). After air drying, the product (28.83 g) was obtained as a yellow solid. H NMR (acetone-d 6 5.20 3H), 7.66-7.75 2H), 7.88-7.92 1H), 8.03-8.09 2H), 8.15 1H), 8.26 2H), 8.48 1H), 8.61-8.68 (m, 2H), 8.80 2H), 9.03 2H).

c. Naphthvl 10-methvlacridan-9-thiocarboxvlate. Naphthyl 10-methylacridinium-9-thiocarboxylate triflate (65.50 g) was suspended in CH Cl (1000 mL) under argon and glacial acetic acid (21.2 ml) and zinc (40.43 g) were added. After stirring overnight, TLC showed the disappearance of the starting material and the formation of a new product. The reaction mixture was filtered through a bed of silica gel and the CH 2 C2 removed in vacuo. The slightly yellow solid obtained was stirred in isopropanol (500 ml), filtered, washed with 500 ml more isopropanol, and allowed to air dry. The pure product (46.36 g) was thereby obtained. H NMR (CDC13) 8 3.47 3H), 5.13 1H), 7.00-7.06 (m, 4H), 7.25-7.49 7H), 7.70-7.79 4H).

d. 9-(Naphthvlthiophohsphorloxvmethlidene) acridan, bis(cyanoethvl) ester. Naphthyl methylacridan-9- thiocarboxylate (17.32 g) in 600 mL of anhydrous THF was added dropwise to a solution of LDA (1.25 eq.) in THF at -78 C. After stirring 90 min at -78 C, the orange-brown solution was treated with a solution of POC1 3 (20.80 g) and pyridine (50 mL) in 150 mL of THF SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 slowly. The dry ice bath was removed after 60 min and stirring continued for 1 h. The solution became brown and formed a precipitate.

The mixture was treated with 3-hydroxypropionitrile (27.8 ml). After stirring over night at room temperature, the precipitated pyridine-HCl was filtered off and washed with THF. The combined filtrates were evaporated in vacuo and the brown oil obtained was separated by column chromatography using 50-100 ethyl acetate/hexanes from which the product was isolated. The yellow oil was dissolved in CH2C1 2 (300 ml), washed with water (450 ml), dried over sodium sulfate, filtered, and dried in vacuo.

This yielded the product (17.76 g) as a yellow solid. 1H NMR (CDCl3) 8 2.33-2.53 4H), 3.53 3H), 3.82-4.06 4H), 6.93 1H), 7.03 1H), 7.10-7.18 2H), 7.25-7.55 5H), 7.79-7.92 5H), 8.02 31 P NMR (CDC1 6 -9.69 (rel. to ext. H PO e. 9-(Naphthvlthiophosphorloxymethvlidene)-10-methylacridan, disodium salt A solution of the bis(cyanoethyl) phosphate compound (17.28 g) in 200 mL of acetone was purged with argon. A solution of NaOH (2.76 g) in 50 ml of water was added and the solution was stirred under argon over night. The precipitate which had formed was suction filtered washed with 20 water in acetone (300 ml) and dried under vacuum. The product (15.07 g) was obtained as a light yellow solid. 1H NMR (DO2) 6 3.22 (s, 3H), 6.67 1H), 6.87 1H), 7.01 1H), 7.08-7.15 51 SUBSTITUTE SHEET (RULE 26) WO 99/40161 WO 9940161PCTIUS98/1 1489 (in, 2H), 7.23 11H), 7. 31-44 3H) 7 .51 Cs, 1H) 7. 59 Cd, 2H), 7. 73 Cd, 1H), 7. 86 1H) 8. 25 1H) 31 p NMR (D 2 8 1.30 Example 5. Additional H-eterocyclic Enol Phospohates Prelpared. The following compounds of formula IV have also been prepared and function in the chemiluminescent reactions of the present invention.

Na 2

O

3 ZAr R1 R 1 8 RR11R2 1

R

1 6 N R 13

R

15 Cl-I 3

R'

4 ComTpound R_ R"Z Ar all H 0 6 R 1 3 OCH 3 0 phenyl 7 R Cl 0 2,6-dimethyiphenyl 8 R-5R 16= 0 phenyl

-CH=CH-CH=CH-

9 all H 0 10 all H S 4-fluorophenyl 11 all H S 4-methoxyphenyl 12 all H S 2,6-dimethylphenyl 12 17 13 R ,R =F S phenyl 14 all H S trifluoroethyl R 11 R 18are H unless otherwise indicated. Compounds 6-8 were obtained as a mixture of double bond isomers.

Each of compounds 5-14 were prepared by the general synthetic methods described in Examples 1-4. In addition 52 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 compounds of the formulas: Na 2 0 3 PO

V

N

I N

CH

3 wherein V is t-butyl, CH 3

OCH

3 F, Cl, Br, I, COCH CN and NO as well as compounds of the formula: Na 2 03PO S

N

CH

3 wherein U is p-I, p-CH 3 m-OCH 3 o-Cl, m-C1, o-Br, m-Br, p- Br and p-NO 2 as well as compounds of this formula having a 3,4-dichloro-, 2,5-dichloro- and 2,6-dichlorophenyl group have been prepared and produce chemiluminescence when reacted with a dihydroxyaromatic compound.

Example 6. Preparation of Protected Dihvdroxvaromatic Compound 4-Hvdroxyhenvl phosphate, disodium salt.

Hydroquinone was protected as the monobenzoate ester in 62 yield by reaction with benzoyl chloride and pyridine in CH2CI 2 at 0

O

C. The ester was phosphorylated with excess POC1 3 in pyridine at 0 OC. The dichlorophosphate derivative was reacted with 10 eq. of 3- 53 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/ 1489 hydroxypropionitrile to produce the bis(cyanoethyl phosphate) in 89 yield. The cyanoethyl and benzoyl protecting groups were hydrolyzed with aq. NaOH/acetone at room temperature over night. The precipitated product was dissolved in methanol and precipitated with acetone to produce 4-hydroxyphenyl phosphate as the disodium salt. H NMR (D20) 6 6.56 1H), 6.92 1H) Example 7. Preparation of Protected Dihvdroxvaromatic Compound 2-Hvdroxr-henvl phosphate, disodium salt.

Catechol was protected as the monobenzoate ester in 62 yield by reaction with benzoyl chloride and pyridine in CH Cl 2 at 0 OC. The ester was phosphorylated with excess POC1 3 and pyridine in CH 2

C

2 at 0 oC. The dichlorophosphate derivative was reacted with 10 eq. of 3hydroxypropionitrile to produce the bis(cyanoethyl phosphate) in 90 yield. The cyanoethyl and benzoyl protecting groups were hydrolyzed with aq. NaOH/acetone at room temperature over night. The precipitated product was dissolved in methanol, precipitated with acetone and the solid washed again with methanol to produce 2hydroxyphenyl phosphate as the disodium salt. H NMR (D 0) 6 6.56-6.61 1H), 6.68-6.71 1H), 6.82-6.87 1H), 7.19-7.22 1H).

Example 8. Preparation of Protected Dihvdroxvaromatic Compound 4-Hvdroxv-2.3.5,6-tetrafluorophenyl phosphate, disodium salt. 2,3,5,6-Tetrafluorohydroquinone was 54 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 protected as the mono(t-butyldimethylsilyl) ether in 46 yield by reaction with t-butyldimethylsilyl chloride and imidazole in DMF over night at room temperature. After chromatographic purification (10 ethyl acetate/hexane), the silyl ether was phosphorylated with excess POC1 in pyridine at 0 The dichlorophosphate derivative was reacted with 10 eq. of 3-hydroxypropionitrile to produce the bis(cyanoethyl phosphate) derivative which had also been desilylated in 55 yield after column chromatography (75-100% ethyl acetate in hexane). The cyanoethyl protecting group was hydrolyzed with aq. NaOH/acetone at room temperature for 2 days. The precipitated product was dissolved in methanol and precipitated with acetone to produce the disodium phosphate salt. 31 P NIR (CD OD) 6 4.244; 19F NMR (CD OD) 6 -169.02 2F), -161.16 2F).

Example 9. Preparation of Protected Dihvdroxvaromatic Compounds 2- and 3-Chloro-4-hvdroxvohenvl phosphate, disodium salt. 2-Chlorohydroquinone, 20 g (Aldrich, Milwaukee, Wisconsin) was recrystallized by dissolving in 350 mL of warm 10% ethyl acetate/CH 2 C12, filtered and added to 1.5 L of hexane. After cooling to 4 oC over night, 11 g of off white solid was collected. The purified 2chlorohydroquinone was monoacetylated with acetyl chloride pyridine in CH 2 C1 2 at ice temperature to produce a mixture of the isomeric monoacetates and the diacetate.

The mixture was phosphorylated with excess POC1 in 3 pyridine at 0 OC. The reaction product was reacted with SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 excess 2-cyanoethanol to produce a 4:1 mixture of isomeric bis(cyanoethyl phosphate) derivatives which were separated from the diacetate by column chromatography (30 ethyl acetate/hexane). The acetate and cyanoethyl protecting groups were hydrolyzed with aq. NaOH/acetone (3 eq. of NaOH) at 0 OC for 19 h. The precipitated product was dissolved in methanol and precipitated with acetone to produce the isomeric disodium phosphate salts. H NMR 6 6.56-7.19 3H); 31p NMR (D20) 6 1.36, 1.39.

Example 10. Preparation of Protected Dihvdroxvaromatic Compound 4-Hvdroxvyhenvl 9-D-calactopyranoside.

Hydroquinone was reacted with a-bromogalactose tetraacetate (1.5 eq.) in ethanol/aq. NaOH at room temperature for 2 days in a light-shielded vessel. Sodium ethoxide (5 eq.) was added and the solution concentrated after one hour. The residue was partitioned between water and ethyl acetate. The water layer was evaporated and the product purified chromatopgraphically (30% methanol/CHCl 2 on silica. H NMR (CD3OD) 6 3.57-3.88 6H), 4.68 (d, 1H), 6.68 2H), 6.96 2H).

Example 11. Preparation of Protected Dihydroxvaromatic Compound 4-Hvdroxyvhenvl g-D-alucuronide. Hydroquinone g) was converted to the monobenzoate ester by reaction with benzoyl chloride and pyridine in CH Cl The yield was 7 g of the ester. IH NMR (CDC1 6 4.880 1H), 6.87 (d, 2H), 7.08 2H), 7.52 2H), 7.64 1H), 8.20 (d, 56 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/1489 2H).

The latter (200 mg) was deprotonated with aq. NaOH in acetone and converted to the 9-glucuronide ethyl ester derivative by reaction with ethyl a-D-bromoglucuronate in absolute ethanol.Purification by prep. TLC afforded 50 mg of the doubly protected compound. 1H NMR (CD OD) 6 1.290 3H), 3.601-3.780 3H), 4.146-4.258 3H), 4.64 (br s, 2H), 4.86 (br s, 1H), 5.192 1H), 7.187-7.285 4H), 7.635 2H), 7.767 1H), 8.208 2H).

A 50 mg sample of the latter compound was hydrolyzed with aq. NaOH in acetone to saponify the benzoate ester and ethyl ester groups. The precipitated product (16 mg) was filtered and dried. H NMR (CD3OD) 6 3.4-3.5 3H), 3.64 1H), 4.65 1H), 6.53 2H), 6.87 2H).

Example 12. Evaluation of Dihydroxvaromatic compounds.

Several dihydroxyaromatic compounds were screened to determine their general effectiveness in inducing the chemiluminescent reaction of 1. Stock solutions of the test compounds (2 x 10 4 M in ethanol/10 DMSO or DMSO) were prepared. A 5 gL aliquot of each was placed into a well of a 96 well microplate. To each well 95 gL of a solution containing 0.66 mM compound 1, in 0.2 M 2-methyl- 2-amino-l-propanol buffer, pH 9.6, 0.88 mM Mg 2 and 0.1 SDS was added. Light intensity was scanned in all wells for 30 min. The maximum intensity and time to maximum were determined for each test well.

57 SUBSTITUTE SHEET (RULE 26) WO 99/40161 WO 9940161PCTIUS98/1 1489 Dihydroxyaromati c comnoound Time to~ 2 -Chiorohydroquinone (DHA- 1) Hydroquinone (DHA-2) 2-Phenyihydroquinone (DHA-3) 3,3,3' 3 '-Tetramethyl-1,1'-spirobisindane- 5,5',6,6'-tetrol (DHA-4) 2,3,5, 6-Tetrafluorohydroquinone (DHA-5) 2-Methyihydroquinone (DHA-6) 2-Methoxyhydroquinone (DHA-7) Catechol (DHA-8) 1, 2-Dihydroxyanthracene (DHA-9) 7, 8-Dihydroxy-6-methoxycounarin (DHA-10) 1-Methyl-6, 7-dihydroxy-1, 2,3,4tetrahydroisoquinoline (DNA-il) 4-Aminoresorcinol (DHA-12) 1,4-Dihydroxynaphthalene (DHA-13) 9, 10-Bis (4-hydroxyphenyl) anthracene (DHA-14) 1,2, 4-Trihydroxybenzene (DHA-15) 4-Bromoresorcinol (DHA-1G) 2, 3-Dihydro-9, 10-dihydroxy-l, 4 -anthraquinone (DHA-17) Ellagic acid (DHA-iS) 2, 5-Dihydroxybenzaldehyde (DH-A--19) 2, 3-Dichloro-5, 8-dihydroxy-l, 4 -naphthoquinone max 6 6 7.5 !3 0 5 15 5 30 7 30 >30 Max S/B >32, 000 20, 000 10,200 3, 000 2, 800 800 480 260 160 150 3 0 !30 !30 7 9 2 Examp~le 13. Kinetic Profile of hei nes e Intensit The rapid generation of chemiluminescence from 58 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 a composition containing the acridan phosphate 1 and hydroquinone (DHA-2) is shown in Figure 1. The reaction mixture comprised 0.66 mM acridan phosphate 1 and 4 M hydroquinone in 2 -methyl-2-amino-l-propanol (221) buffer, 0.2 M, pH 9.6 also containing 0.88 mM MgCl Example 14. Chemiluminescent Detection of Acridan Phosphate 2 with DHA-1. The following example illustrates the ability to detect very small quantities of a heterocyclic enol phosphate compound by use of the chemiluminescent reaction with a dihydroxyaromatic compound.

Into each of 3 white microwells was placed a 100 gL aliquot of a solution of acridan phosphate 2 (dilutions contained between 6.6 x 10 11 and 6.6 x 10 7 mol) in 0.2 M 221 buffer, pH 9.6, 0.88 mM MgCl2, 0.1 SDS. A 100 tiL aliquot of a 0.01 mM solution of 2-chlorohydroquinone (DHA-1) in water was injected into each well and light intensity was measured at 2.5 min. The results shown in Figure 2 show that light intensity is a direct function of the quantity of the phosphate compound over the entire range tested.

This example demonstrates that the heterocyclic enol phosphate can be detected at concentrations low enough that a suitably functionalized derivative with a linkable group can function as a signal-generating label in a specific binding assay.

59 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCTIUS98/11489 Example 15. Reagent for Chemiluminescent Detection of Alkaline Phosphatase. An effective reagent composition for chemiluminescent detection of alkaline phosphatase comprised 0.2 M 221 buffer, pH 9.6, 0.88 mM MgC1 2 0.33 mM acridan phosphate 1, and 1 mM 4-hydroxyphenyl phosphate.

Reaction of 100 L of this composition with 8 x 10 17 mol of AP at 25 oC in a test tube housed in a Turner luminometer produced easily measurable blue chemiluminescence. The time profile of a typical reaction is shown in Figure 3.

Example 16. Chemiluminescent Detection with Other Heterocvclic Enol Phosphate Compounds. In the manner of the previous Example, compositions containing each of acridan phosphates 2-4 in place of compound 1 were reacted with AP at 25 oC. Each produced easily measurable chemiluminescence discernable above the background in the absence of AP.

Example 17. Effect of Concentration of Protected Dihvdroxvaromatic Compound. A study was performed to determine the range of concentrations of protected dihydroxyaromatic compound useful in chemiluminescent assays of a hydrolytic enzyme. A reagent consisting of a 0.33 mM solution of acridan phosphate 1 and 0.88 mM MgC1 in 0.2 M 221 buffer, pH 9.6 containing concentrations of 4-hydroxyphenyl phosphate varying from 0 to 10 mM was SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 reacted with 8 x 10 1 mol of AP at 25 °C (in mg/mL: a, 1; b, 3.3; c, 0.66; d, 0.83; e, 10; f, 0.1; g, Figure 4 depicts the various kinetic profiles of chemiluminescence emission. The fact that the solution lacking 4hydroxyphenyl phosphate produced almost no chemiluminescence demonstrates that light emission is not due simply to removal of the phosphate protecting group from 1 by AP followed by autoxidation of the enolate.

Example 18. Linearity of Detection of AP with Acridan Phosphate 1 in a One Step Assay. A chemiluminescent assay of AP was performed by reacting varying quantities of AP with 100 4L portions of a reagent composition comprising 0.66 mM acridan phosphate 1, 0.66 mM 4-hydroxyphenyl phosphate and 0.88 mM MgC1 2 in 0.2 M 221 buffer, pH 9.6 at OC placed in a 96-well plate housed in a Labsystems Luminoskan luminometer. Light intensity measured at 25 min correlated with amount of enzyme in the range 8 x 10 17 8 x 10 mol as shown in Figure Example 19. Chemiluminescent Detection of i-Galactosidase.

A stock solution of 9-galactosidase (Grade VIII, cat. No.

G 5635, Sigma Chemical Co.) was serially diluted in 0.01 M phosphate buffer, pH 7.0, 0.01 M NaCI, 0.01 M MgC12 to contain between 4.9 x 10 15 and 1.6 x 10 18 mol of enzyme/pL. 130 iL Aliquots of a 1 mM solution of 4hydroxyphenyl-g-D-galactoside in 0.05 M phosphate buffer, pH 7.5, 0.01 M NaCI, 3 mM MgC12 were incubated with 2.6 gL 61 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 of each the enzyme dilutions at 37 0 C for 35 min. Duplicate ~LL portions were transferred into black microwell strips and cooled to room temperature. A 50 gL portion of a solution of acridan phosphate 3 having the following composition: 0.2 M 221 buffer, pH 9.6, 0.88 mM MgC 2, 0.66 mM acridan phosphate 3, and 0.1 SDS was injected into each well. The amount of enzyme in the wells ranged from 4.9 x 10 15 and 1.6 x 10 18 mol of E-galactosidase.

Chemiluminescence intensity measured at 5 min in relation to the amount of I-galactosidase is shown in Figure 6.

Example 20. Chemiluminescent Detection of 9-Glucuronidase.

A stock solution of 9-glucuronidase (type X-A, cat. No. G 7896, Sigma Chemical Co., MW 290,000) was serially diluted in 0.01 M phosphate buffer, pH 7.0, 0.15 M NaCl to contain between 3.3 x 10 14 and 3.3 x 10 1 mol of enzyme/gL. 130 plL Aliquots of a 1 mM solution of 4hydroxyphenyl--D-glucuronide in 0.05 M phosphate buffer, pH 7.0, 0.01 M NaC1 were incubated with 2.6 JLL of each the enzyme dilutions at 37 0 C for 30 min. Duplicate 50 LL portions were transferred into black microwell strips and cooled to room temperature. A 50 portion of a solution of acridan phosphate 4 having the following composition: 0.2 M 221 buffer, pH 9.6, 0.88 mM MgCl 2 0.66 mM acridan phosphate 4, and 0.1 SDS was injected into each well.

Chemiluminescence intensity measured at 11 min in relation to the amount of f-glucuronidase is shown in Figure 7.

62 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 ExamDle 21. Western Blot Assay using an Alkaline PhosDhatase-Coniuate. Compositions of the present invention were used to detect and quantify a protein antigen, 9-galactosidase in a Western blot with an APlabeled antibody on a nitrocellulose membrane. Dilutions of i-galactosidase containing from 5000, 1000, 180, 30 and pg, respectively, of protein were electrophoresed and transferred to nitrocellulose membranes (Hybond ECL, Amersham, Arlington Heights, Illinois). The membranes were blocked with 1% non-fat milk (NFM) in T-TBS (0.05% Tween in TBS; TBS is 50 mmol/L Tris-HCl, pH 7.4, 0.15 mol/L NaC1) for 1 h at room temperature and then treated sequentially with a 1:1500 dilution of a 3.3 Jg/mL solution of mouse anti-i-galactosidase in blocking buffer, T-TBS wash buffer and then a 1:600 dilution of a 416 mU/mL solution of sheep anti-mouse IgG-AP conjugate. After washing with T-TBS, the membranes were soaked briefly with a detection reagent comprising 0.2 M 221 buffer, pH 9.6 containing 0.88 mM MgCl2, 0.66 mM hydroquinone phosphate disodium salt, 0.66 mM acridan phosphate 3 and 0.1 SDS.

The membranes were placed between transparent plastic sheets and exposed to X-ray film. All five bands of 9galactosidase were detected after 15 min with a 1 min exposure. The light produced using these compositions led to intense emission which could be imaged for at least 1 day.

63 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 Example 22. Dot Blot Assay. A representative example of the use of a reagent of the present invention in a dot blot assay is demonstrated in the following example.

Digoxigenin-labeled DNA (pBR328), anti-digoxigenin-AP conjugate were supplied as a kit (Genius 3, Boehringer- Mannheim, Indianapolis, Indiana), blocking buffer and positively charged nylon membrane were obtained from Boehringer-Mannheim. The wash buffer was 0.1 M maleic acid, pH 7.5, 0.15 M NaC1, 0.05 SDS. The detection reagent comprised 0.66 mM acridan phosphate 2, 0.66 mM 4hydroxyphenyl phosphate, 0.88 mM MgCl 2 0.1 SDS in 0.2 M 221 buffer, pH 9.6.

DNA dilutions (10, 3, 1, 0.3, 0.1, 0.03, 0.01 pg) were dot blotted onto nylon membranes, blocked, washed and reacted with antibody-AP conjugate according to manufacturer's protocol. Excess buffer was drained off and blots soaked in a detection reagent described in for 3 min. Excess reagent was drained off, and the blots placed between transparent sheets and exposed to X-ray film for varying lengths of time. An immediate 1 min exposure detected the 10 pg 0.3 pg spots. After 30 min, all seven spots were detected with a 20 min exposure. Multiple exposures could be performed for several days. For example, after 5 days, all seven spots were detected with a 1-5 min exposure. Detection reagents containing 0.66 mM acridan phosphate 3, or 0.66 mM solution of a mixture of 2- and 3 -chloro-4-hydroxyphenyl phosphate produced comparable results.

64 SUBSTITUTE SHEET (RULE 26) P:\WPDOCS\Hjw\pec\7499280pgs.doc-09/1 1/01 The foregoing description and examples are illustrative only and not to be considered as restrictive. It is to be recognised that modifications of the specific compounds and methods not specifically disclosed can be made without departing from the spirit and scope of the present invention which is limited only by the appended claims.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the 10 common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or :o "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not .the exclusion of any other integer or step or group of S. integers or steps.

S

o*o

Claims

1. A method of generating chemiluminescence comprising reacting in the presence of oxygen a) a dihydroxyaromatic compound which comprises from 1- carbocyclic aromatic rings and which is substituted with two hydroxy groups separated by an even number of ring carbon atoms; and b) a heterocyclic enol phosphate compound having the formula MnO 3 PO V ZR 10 R 1 8 R 11 1 R 16 N R 13 R 1 5 R19 R 14 wherein R 10 is an organic group containing up to 50 non- hydrogen atoms selected from C, N, 0, S, P and halogen atoms, each of R 1 1 R 18 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, alkenyl, alkynyl, alkoxy, aryloxy, halogen, amino, substituted amino, carboxyl, carboalkoxy, carboxamide, cyano, and sulfonate groups, and wherein pairs of adjacent groups can complete a benzo-fused ring, R 19 is an organic group containing up to 50 non- hydrogen atoms selected from C, N, O, S, P and halogen atoms, Z is selected from O and S atoms, each M is independently selected from H and a cationic center and n is a number which satisfies electroneutrality; and provided that any one of R 11 R 18 or a substituent on any one of R 10 66 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 R 19 can be a group -A-Q wherein A is a spacer group selected from C 1 -C 0 l alkylene and C 2 -C 10 oxyalkylene groups and Q is a linking group capable of forming a covalent bond selected from halogen, diazo, -NCO, -NCS, -CHO, acid anhydride, oxiranyl, succinimidoxycarbonyl, maleimide, cyano, triazole, tetrazole, hydroxyl, -COOH, thiol, primary amino and secondary amino groups.

2. The method of Claim 1 wherein the dihydroxyaromatic compound comprises an aromatic ring system selected from benzene, biphenyl, naphthalene, and anthracene rings.

3. The method of Claim 1 wherein the dihydroxyaromatic compound has the formula I OH R 1 R 4 R 2 R 3 I wherein at least one of R 1 and R 3 is an OH group, the other one of R 1 or R 3 and R 2 R 4 and R 5 are each independently selected from hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, amino, aminoalkyl, carboxyl -C(=O)OH, carboxyl ester -C(=0)OR 6 formyl alkylcarboxy OC(=O)R 6 arylcarboxy -OC(=O)R 9 and halogen groups, pairs of adjacent groups, when taken together, can complete a five or six-membered aliphatic or aromatic ring, R 6 is a lower alkyl group, R 7 and R 8 are each H or a lower alkyl group and R 9 is an aryl ring group. 67 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

4. The method of Claim 3 wherein the group R 3 in the compound of formula I is the OH group and R 1 R 2 R 4 and R are independently selected from hydrogen, alkyl, alkoxy, halogen, aryl and aralkyl groups. The method of Claim 3 wherein the group R 3 in the compound of formula I is the OH group, R 2 R 4 and R 5 are each hydrogen and wherein R 1 is selected from hydrogen, alkyl, alkoxy, halogen, aryl and aralkyl groups.

6. The method of Claim 1 wherein the group R 19 in the heterocyclic enol phosphate compound is selected from substituted and unsubstituted lower alkyl groups and substituted and unsubstituted benzyl groups.

7. The method of Claim 1 wherein the group R 10 in the heterocyclic enol phosphate compound is selected from alkyl, substituted alkyl, aryl, substituted aryl, aralkyl and substituted aralkyl groups. 68 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

8. The method of Claim 1 wherein the heterocyclic enol phosphate compound has the formula Na 2 O 3 PO ZAr R 18 R 1 R 17 8 R12 R 16 N R13 19 R4 wherein Z is O or S, Ar is selected from a phenyl group, a substituted phenyl group and a naphthyl group, R 19 is a lower alkyl group and each of R 11 to R 18 is independently selected from hydrogen and lower alkoxy groups.

9. The method of Claim 5 wherein the heterocyclic enol phosphate compound has the formula Na 2 0 3 PO. ZAr R18 R 1 RI7 R 1 2 R 1 5 19 R 1 4 wherein Z is 0 or S, Ar is selected from a phenyl group, a substituted phenyl group and a naphthyl group, R 19 is a lower alkyl group and each of R11 to R 18 is independently selected from hydrogen and lower alkoxy groups. 69 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 The method of Claim 1 wherein the heterocyclic enol phosphate compound is selected from Na 2 O 3 PO OPh Na 2 O 3 PO SPh Na 2 O 3 PO SG N N N CH 3 CH 3 CH 3 and Na 2 3 PO

11-. S N CH 3 11. The method of Claim 5 wherein the heterocyclic enol phosphate compound is selected from Na 2 O 3 PO V. OPh Na 2 O 3 PO V.NSPh Na 2 O 3 PO S-0S a N N N II I CH 3 CH 3 CH 3 and Na 2 3 PO. S0 N CH7 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

12. The method of Claim 1 wherein the heterocyclic enol phosphate compound contains the group -A-Q.

13. The method of Claim 8 wherein the heterocyclic enol phosphate compound contains the group -A-Q as a substituent on R 10 or R 19 or at any one of the R 11 R 18 positions.

14.The method of Claim 1 wherein oxygen is supplied dissolved in a solution containing the dihydroxyaromatic compound and heterocyclic enol phosphate compound at equilibrium with the atmosphere. method of Claim 1 wherein the dihydroxyaromatic compound and heterocyclic enol phosphate compound are present in a solution comprising an aqueous buffer having a pH between about 7 and about 10.5, the dihydroxyaromatic compound is present at a concentration of 0.001-20 mM and the heterocyclic enol phosphate compound at a concentration of 0.001-20 mM. 71 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

16. A composition for generating chemiluminescence in the presence of oxygen comprising a) a dihydroxyaromatic compound which comprises from 1- carbocyclic aromatic rings and which is substituted with two hydroxy groups separated by an even number of ring carbon atoms; and b) a heterocyclic enol phosphate compound having the formula MnO 3 PO ZRI 0 Ris R R 1 7 R12 R1 6 N R 1 3 R 1 5 R19 R 1 4 wherein R 10 is an organic group containing up to 50 non- hydrogen atoms selected from C, N, O, S, P and halogen atoms, each of R 11 R 1 8 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, alkenyl, alkynyl, alkoxy, aryloxy, halogen, amino, substituted amino, carboxyl, carboalkoxy, carboxamide, cyano, and sulfonate groups, and wherein pairs of adjacent groups can complete a benzo-fused ring, R 1 9 is an organic group containing up to 50 non- hydrogen atoms selected from C, N, O, S, P and halogen atoms, Z is selected from O and S atoms, each M is independently selected from H and a cationic center and n is a number which satisfies electroneutrality; and provided that any one of R 11 R 18 or a substituent on any one of R 10 R 1 9 can be a group -A-Q wherein A is a spacer group selected from C 1 -Clo alkylene and C 2 -C 10 oxyalkylene groups 72 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 and Q is a linking group capable of forming a covalent bond selected from halogen, diazo, -NCO, -NCS, -CHO, acid anhydride, oxiranyl, succinimidoxycarbonyl, maleimide, cyano, triazole, tetrazole, hydroxyl, -COOH, thiol, primary amino and secondary amino groups. 17 The composition of Claim 16 wherein the dihydroxyaromatic compound comprises an aromatic ring system selected from benzene, biphenyl, naphthalene, and anthracene rings.

18. The composition of Claim 16 wherein the dihydroxyaromatic compound has the formula I OH RS ^^R 1 R 5 R 4 R 2 R 3 I wherein at least one of R 1 and R 3 is an OH group, the other one of R 1 or R 3 and R 2 R 4 and R 5 are each independently selected from hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, amino, aminoalkyl, carboxyl -C(=O)OH, carboxyl ester -C(=0)OR 6 formyl alkylcarboxy OC(=O)R 6 arylcarboxy -OC(=O)R 9 and halogen groups, pairs of adjacent groups, when taken together, can complete a five or six-membered aliphatic or aromatic ring, R 6 is a lower alkyl group, R 7 and R 8 are each H or a lower alkyl group and R 9 is an aryl ring group. 73 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

19. The composition of Claim 18 wherein the group R 3 in the compound of formula I is the OH group and R 1 R 2 R 4 and R are independently selected from hydrogen, alkyl, alkoxy, halogen, aryl and aralkyl groups. The composition of Claim 18 wherein the group R 3 in the compound of formula I is the OH group, R 2 R 4 and R 5 are each hydrogen and R 1 is selected from hydrogen, alkyl, alkoxy, halogen, aryl and aralkyl groups.

21. The composition of Claim 16 wherein the group R 19 in the heterocyclic enol phosphate compound is selected from substituted and unsubstituted lower alkyl groups and substituted and unsubstituted benzyl groups.

22. The composition of Claim 16 wherein the group R 10 in the heterocyclic enol phosphate compound is selected from alkyl, substituted alkyl, aryl, substituted aryl, aralkyl and substituted aralkyl groups. 74 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/1 489

23. The composition of Claim 16 wherein the heterocyclic enol phosphate compound has the formula Na 2 0 3 PO ZAr R 1 8 R 11 R17 R 1 2 R1 6 N R 13 R 1 5 j19 R 14 wherein Z is 0 or S, wherein Ar is selected from a phenyl group, a substituted phenyl group and a naphthyl group, wherein R 1 9 is a lower alkyl group and wherein each of R 11 to R 18 is independently selected from hydrogen and lower alkoxy groups.

24. The composition of Claim 20 wherein the heterocyclic enol phosphate compound has the formula Na 2 0 3 PO ZAr R 18 R1 R17 R 12 R16 N R 1 3 R 1 5 19 R 14 wherein Z is O or S, wherein Ar is selected from a phenyl group, a substituted phenyl group and a naphthyl group, wherein R 1 9 is a lower alkyl group and wherein each of R 1 1 to R 18 is independently selected from hydrogen and lower alkoxy groups. SUBSTITUTE SHEET (RULE 26) WO 99/40161 WO 9940161PCT/US98/1 1489 The composition of Claim 16 wherein the heterocyclic enol phosphate compound is selected from Na 2 0 3 P0,. OPh Na 2 0 3 POI, SPh Na 2 0 3 PO O.S a N N N N N N N I I I UH 3 CH- 3 (ch 3 and Na 2 OJPO,%--,S 1 N

26. The composition of Claim 20 wherein the heterocyclic enol phosphate compound is selected from Na 2 O 3 PONI OPh Na 2 0 3 PO1-. SPh Na 2 3 PO, S0- aC NN N N N 51I I I C'7' 3 c}- 3 cH and Na 03 PON. 0 S I SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

27. The composition of Claim 16 wherein the heterocyclic enol phosphate compound contains the group -A-Q.

28. The composition of Claim 27 wherein the heterocyclic enol phosphate compound contains the group -A-Q as a substituent on R 10 or R 19 or at any one of the R 11 R 18 positions.

29. The composition of Claim 16 comprising an aqueous buffer having a pH in the range 7-10.5, containing the dihydroxyaromatic compound at a concentration of 0.001-20 mM and the heterocyclic enol phosphate compound at a concentration of 0.001-20 mM. The composition of Claim 29 having a pH in the range of 8-10 and containing the dihydroxyaromatic compound at a concentration of about 0.05-5 mM and the heterocyclic enol phosphate compound at a concentration of about 0.05-5 mM.

31. The method of Claim 1 further comprising producing the dihydroxyaromatic compound by reacting a protected dihydroxyaromatic compound in which one hydroxy group of a dihydroxyaromatic compound is protected by an enzyme- cleavable group X with a hydrolytic enzyme to remove the enzyme-cleavable group. 77 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

32. The method of Claim 31 wherein the reaction of the hydrolytic enzyme with the protected dihydroxyaromatic compound is performed in the absence of the heterocyclic enol phosphate.

33. The method of Claim 31 wherein the reaction of the hydrolytic enzyme with the protected dihydroxyaromatic compound is performed in the presence of the heterocyclic enol phosphate.

34. The method of Claim 31 wherein the hydrolytic enzyme is selected from alkaline phosphatase, acid phosphatase, 9-galactosidase, E-glucuronidase and S-glucosidase and the enzyme-cleavable group is selected from phosphate, 9-galactoside, 9-glucuronide and 9-glucoside groups. 78 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 The method of Claim 31 wherein the protected dihydroxyaromatic compound has the formula OH RS ,R 1 R4R 2 R 3 II wherein one of R 1 or R 3 is an OX group, wherein X is the enzyme-cleavable group, the other one of R 1 or R 3 and R 2 R 4 and R 5 are each independently selected from hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, amino, aminoalkyl, carboxyl carboxyl ester -C(=O)OR 6 formyl alkylcarboxy -OC(=O)R 6 arylcarboxy OC(=0)R 9 and halogen groups, pairs of adjacent groups, when taken together, can complete a benzo-fused ring, R 6 is a lower alkyl group, R 7 and R 8 are each H or a lower alkyl group and R 9 is an aryl ring group. 79 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

36. The method of Claim 35 wherein R 3 is the OX group, R 2 R 4 and R 5 are each hydrogen and R 1 is selected from hydrogen, alkyl, alkoxy, halogen, aryl and aralkyl groups.

37. The method of Claim 31 wherein the heterocyclic enol phosphate compound has the formula Na 2 O 3 PO ZAr R 18 R1 R175 R12 R 16 N R13 R 1 5 R19 R4 wherein Z is 0 or S, Ar is selected from a phenyl group, a substituted phenyl group and a naphthyl group, R 19 is a lower alkyl group and each of R 11 to R 1 8 is independently selected from hydrogen and lower alkoxy groups. SUBSTITUTE SHEET (RULE 26) WO 99/40161 WO 99/016 1PCTIUS98/I 1489

38. The method of Claim 31 wherein the heterocyclic enol phosphate compound is selected from Na 2 O 3 PO 1,1 OPh Na 2 O 3 po,.N,,SPh Na2 3 PON N K N cH' 3 cH 3 Ch 3 and Na 2 O 3 3 N 81 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

39. A composition for generating chemiluminescence by reaction with a hydrolytic enzyme comprising: a) a protected dihydroxyaromatic compound which comprises from 1-5 carbocyclic aromatic rings and which is substituted with two hydroxy groups separated by an even number of ring carbon atoms, wherein one hydroxy group is protected by an enzyme-cleavable group X; and b) a heterocyclic enol phosphate compound having the formula MnO 3 PO ZR 10 R 1 8 R 11 R 1 R 1 R 16 N R1 3 R 15 R19 R 14 wherein R 1 0 is an organic group containing up to 50 non- hydrogen atoms selected from C, N, O, S, P and halogen atoms, each of R 11 R 18 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, alkenyl, alkynyl, alkoxy, aryloxy, halogen, amino, substituted amino, carboxyl, carboalkoxy, carboxamide, cyano, and sulfonate groups, and wherein pairs of adjacent groups can complete a benzo-fused ring, R 19 is an organic group containing up to 50 non-hydrogen atoms selected from C, N, O, S, P and halogen atoms, Z is selected from O and S atoms, each M is independently selected from H and a cationic center and n is a number which satisfies electroneutrality. 82 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 The composition of Claim 39 wherein the dihydroxyaromatic compound comprises an aromatic ring system selected from benzene, biphenyl, naphthalene, and anthracene rings.

41. The composition of Claim 39 wherein the protected dihydroxyaromatic compound has the formula I OH R 5 R 1 R R2 R 3 I wherein at least one of R 1 and R 3 is an OX group, the other one of R 1 or R 3 and R 2 R 4 and R 5 are each independently selected from hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, amino, aminoalkyl, carboxyl -C(=O)OH, carboxyl ester -C(=0)OR 6 formyl alkylcarboxy OC(=O)R 6 arylcarboxy -OC(=0)R 9 and halogen groups wherein pairs of adjacent groups, when taken together, can complete a five or six-membered aliphatic or aromatic ring, R 6 is a lower alkyl group, R 7 and R 8 are each H or a lower alkyl group and R 9 is an aryl ring group.

42. The composition of Claim 41 wherein the group R 3 in the compound of formula I is the OX group and R 1 R 2 R 4 and R are independently selected from hydrogen, alkyl, alkoxy, halogen, aryl and aralkyl groups. SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

43. The composition of Claim 41 wherein the group R 3 in the compound of formula I is the OX group, R 2 R 4 and R 5 are each hydrogen and R 1 is selected from hydrogen, alkyl, alkoxy, halogen, aryl and aralkyl groups.

44. The composition of Claim 39 wherein the group R 19 in the heterocyclic enol phosphate compound is selected from substituted and unsubstituted lower alkyl groups and substituted and unsubstituted benzyl groups. The composition of Claim 39 wherein the group R 10 in the heterocyclic enol phosphate compound is selected from alkyl, substituted alkyl, aryl, substituted aryl, aralkyl and substituted aralkyl groups.

46. The composition of Claim 39 wherein the heterocyclic enol phosphate compound has the formula Na203POO ZAr R 1 8 R 1 R17 R18 R 12 R 16 N R13 R 15 19 R 14 wherein Z is 0 or S, Ar is selected from a phenyl group, a substituted phenyl group and a naphthyl group, R 19 is a lower alkyl group and each of R 11 to R 18 is independently selected from hydrogen and lower alkoxy groups. 84 SUBSTITUTE SHEET (RULE 26) WO 99/40161 WO 99/016 1PCTIUS98/1 1489

47. The composition of Claim 41 wherein the heterocyclic enol phosphate compound has the formula Na 2 O 3 PO1,1. ZAr R4 18 R~ 1 1 RO R 12 16 NJ R 13 R 15 1 R4 wherein Z is 0 or S, Ar is selected from a phenyl group, a substituted phenyl group and a naphthyl group, R 1 9 is a lower alkyl group and each of R 11 to R 18 is independently selected from hydrogen and lower alkoxy groups.

48. The composition of Claim 39 wherein the heterocyclic enol phosphate compound is selected from Na 2 O 3 Na 2 O 3 PO,--,,SPh Na 2 O 3 PO. -0 N N N N II. I :N N N I 1 1 cl- 3 C 3 cH t 3 and NaAOPONI.S-C o N CH' 3 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

49. The composition of Claim 39 comprising an aqueous buffer having a pH in the range 7-10.5, containing the protected dihydroxyaromatic compound at a concentration of 0.001-20 mM and the heterocyclic enol phosphate compound at a concentration of 0.001-20 mM. The composition of Claim 49 having a pH in the range of 8-10 and containing the protected dihydroxyaromatic compound at a concentration of about 0.05-5 mM and the heterocyclic enol phosphate compound at a concentration of about 0.05-5 mM.

51. The composition of Claim 49 additionally comprising an anionic surfactant.

52. The composition of Claim 51 wherein the anionic surfactant is a C 10 -C 20 alkyl sulfate.

53. The composition of Claim 51 wherein the anionic surfactant is sodium dodecyl sulfate. 86 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

54. A method of conducting an assay of an analyte comprising: a) reacting in the presence of oxygen a dihydroxyaromatic compound which comprises from carbocyclic aromatic rings and which is substituted with two hydroxy groups separated by an even number of ring carbon atoms with a heterocyclic enol phosphate compound to produce chemiluminescence wherein the heterocyclic enol phosphate compound has the formula Mn03PO V ZR 10 R 18 I R R17 R12 R16 N R 13 R 1 5 I 19 R 14 wherein R 1 0 is an organic group containing up to 50 non- hydrogen atoms selected from C, N, O, S, P and halogen atoms, each of R 11 R 18 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, alkenyl, alkynyl, alkoxy, aryloxy, halogen, amino, substituted amino, carboxyl, carboalkoxy, carboxamide, cyano, and sulfonate groups, and wherein pairs of adjacent groups can complete a benzo-fused ring, R 19 is an organic group containing up to 50 non-hydrogen atoms selected from C, N, O, S, P and halogen atoms, Z is selected from O and S atoms, each M is independently selected from H and a cationic center and n is a number which satisfies electroneutrality; and 87 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489 provided that any one of R 11 R 18 or a substituent on any one of R 10 R 19 can be a group -A-Q wherein A is a spacer group selected from C 1 -C 1 0 alkylene and C 2 -C 1 0 oxyalkylene groups and Q is a linking group capable of forming a covalent bond selected from halogen, diazo, -NCO, -NCS, CHO, acid anhydride, oxiranyl, succinimidoxycarbonyl, maleimide, cyano, triazole, tetrazole, hydroxyl, -COOH, thiol, primary amino and secondary amino groups; b) measuring the chemiluminescence; and c) relating the chemiluminescence to the amount of the analyte. The method of Claim 54 wherein the dihydroxyaromatic compound comprises an aromatic ring system selected from benzene, biphenyl, naphthalene, and anthracene rings. 88 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

56. The method of Claim 54 wherein the dihydroxyaromatic compound has the formula I OH R1 R4 R2 R 3 I wherein at least one of R 1 and R 3 is an OH group, the other one of R 1 or R 3 and R 2 R 4 and R 5 are each independently selected from hydrogen, alkyl, alkoxy, alkenyl, alkynyl, aryl, aralkyl, amino, aminoalkyl, carboxyl -C(=O)OH, carboxyl ester -C(=O)OR 6 formyl alkylcarboxy OC(=0)R 6 arylcarboxy -OC(=0)R 9 and halogen groups, pairs of adjacent groups, when taken together, can complete a five or six-membered aliphatic or aromatic ring, R 6 is a lower alkyl group, R 7 and R 8 are each H or a lower alkyl group and R 9 is an aryl ring group.

57. The method of Claim 56 wherein the group R 3 in the compound of formula I is the OH group and R 1 R 2 R 4 and R are independently selected from hydrogen, alkyl, alkoxy, halogen, aryl and aralkyl groups.

58. The method of Claim 56 wherein the group R 3 in the compound of formula I is the OH group, R 2 R 4 and R 5 are hydrogen and R 1 is selected from hydrogen, alkyl, alkoxy, halogen, aryl and aralkyl groups. 89 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

59. The method of Claim 54 wherein the group R 19 in the heterocyclic enol phosphate compound is selected from substituted and unsubstituted lower alkyl groups and substituted and unsubstituted benzyl groups. The method of Claim 54 wherein the group R 1 0 in the heterocyclic enol phosphate compound is selected from alkyl, substituted alkyl, aryl, substituted aryl, aralkyl and substituted aralkyl groups.

61. The method of Claim 54 wherein the heterocyclic enol phosphate compound has the formula Na 2 0 3 PO ZAr R18 R" R 17 R12 I R6N R/ 13 19 R 14 wherein Z is O or S, Ar is selected from a phenyl group, a substituted phenyl group and a naphthyl group, R 19 is a lower alkyl group and each of R 11 to R 18 is independently selected from hydrogen and lower alkoxy groups. SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

62. The method of Claim 56 wherein the heterocyclic enol phosphate compound has the formula Na203PO ZAr R 1 8 R 11 R 17 R 12 I 1 R16 N R 13 R 1 5 19 R 1 4 wherein Z is O or S, Ar is selected from a phenyl group, a substituted phenyl group and a naphthyl group, R 19 is a lower alkyl group and each of R 11 to R 1 8 is independently selected from hydrogen and lower alkoxy groups.

63. The method of Claim 54 wherein the heterocyclic enol phosphate compound is selected from 3 PO OPh Na 2 0 3 PO SPh Na 2 0 3 PO S- C I I I CH 3 CH 3 CH 3 and Na 2 0 3 PO, S N I CH 3 91 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

64. The method of Claim 56 wherein the heterocyclic enol phosphate compound is selected from Na 2 0 3 PO OPh Na 2 0 3 PO SPh Na 2 0 3 PO S- I I I CH 3 CH 3 CH 3 and Na 2 O 3 PO, S N CH 3 The method of Claim 54 wherein the heterocyclic enol phosphate compound contains the group -A-Q and further comprising; reacting the heterocyclic enol phosphate compound with a specific binding pair member to form a detectably labeled specific binding pair member; contacting the labeled specific binding pair member with its specific binding partner to form a labeled specific binding pair; and contacting the labeled specific binding pair with the dihydroxyaromatic compound to produce chemiluminescence for detecting the analyte. 92 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/11489

66. The method of Claim 65 wherein the heterocyclic enol phosphate compound contains the group -A-Q as a substituent on R 1 0 or R 19 or at any one of the R 1 R 18 positions.

67. The method of Claim 54 further comprising the step of reacting a protected dihydroxyaromatic compound in which one hydroxy group of a dihydroxyaromatic compound is protected with an enzyme-cleavable group X with a hydrolytic enzyme to produce the dihydroxyaromatic compound.

68. The method of Claim 67 wherein the reaction of the hydrolytic enzyme with the protected dihydroxyaromatic compound is performed in the absence of the heterocyclic enol phosphate.

69. The method of Claim 67 wherein the reaction of the hydrolytic enzyme with the protected dihydroxyaromatic compound is performed in the presence of the heterocyclic enol phosphate. The method of Claim 67 wherein the hydrolytic enzyme is selected from alkaline phosphatase, acid phosphatase, E-galactosidase, 1 -glucuronidase and 9-glucosidase and the enzyme-cleavable group is selected from phosphate, E-galactoside, 9-glucuronide and 9-glucoside groups. 93 SUBSTITUTE SHEET (RULE 26) WO 99/40161 PCT/US98/1 489

71. The method of Claim 67 wherein the analyte is the hydrolytic enzyme.

72. The method of Claim 67 wherein the hydrolytic enzyme is provided as a label on a specific binding pair member.

73. A compound which is 3-chloro-4-hydroxyphenyl phosphate, disodium salt having the formula OH C1 OPO 3 Na 2

74. A compound which is 2-chloro-4-hydroxyphenyl phosphate, disodium salt having the formula OH OPO 3 Na 2 A compound which is 2 ,3,5,6-tetrafluoro-4-hydroxyphenyl phosphate, disodium salt having the formula OH F F F F OPO 3 Na 2 94 SUBSTITUTE SHEET (RULE 26) P:AWPDOCS\Hjvqp-\749920pg dOC-09/1 1/01

76. A method of generating chemiluminescence substantially as herein described with reference to the Examples.

77. Compositions for generating chemiluminescence substantially as herein described with reference to the Examples.

78. A method of conducting an assay of an analyte substantially as herein described with reference to the 10 Examples. S. DATED this 9th day of November 2001 LUMIGEN INC. By its Patent Attorneys DAVIES COLLISON CAVE 0